Method for transdifferentiating non-neuronal cells into neurons and application

Abstract

There is provided a method of transdifferentiating a non-neuronal cell of a mammal into neurons, comprising: providing a negative modulator capable of reducing expression of a negative regulatory gene, the negative regulatory gene comprising RCOR1, RCOR2, RCOR3, Sin3a, Sin3b, HDAC1, HDAC2, KDM1A, PHF21A, BAF53a, G9a, USP14, HuR, BrG1, EZH2, CDYL, or HMG20B, and / or providing a positive modulator capable of increasing expression of a positive regulatory gene, the positive regulatory gene comprising DPYSL2, BAF45b, SCF, HuB, HuC, HuD, CYP1B1, or BTRC, the negative regulatory gene comprising RCOR1, RCOR2, RCOR3, Sin3a, Sin3b, an effective amount of the negative regulator or the positive regulator is contacted with the non-neuronal cells, so that the non-neuronal animal cells can be induced to be transdifferentiated into neurons.

Description

Methods and applications of transdifferentiation of non-neuronal cells into neurons

[0001] This application claims priority to patent application number PCT / CN2022 / 115708 filed on August 30, 2022. Technical Field

[0002] The present application relates to the field of translational medicine, and more specifically, to a method for transdifferentiating non-neuronal cells into neurons, and the use of the method for preparing drugs for treating or preventing diseases related to neuronal damage or neuronal death. Background Art

[0003] The primary pathological changes caused by nervous system damage and various neurodegenerative diseases are neuronal degeneration, necrosis, and neuronal damage. Because the nervous system (e.g., brain, spinal cord, optic nerve, etc.) has very limited self-repair capabilities and is difficult to repair, diseases associated with neuronal dysfunction or death have always been difficult to treat. These include Parkinson's disease, schizophrenia, depression, Alzheimer's disease, Huntington's disease, sleep disorders, brain trauma, stroke, visual system diseases associated with RGC or photoreceptor cell dysfunction or death, blindness, and deafness. Current treatments only mitigate disease progression, and effective treatments are still lacking.

[0004] Cell transdifferentiation refers to the process by which one type of differentiated cell, under certain conditions, can transform structurally and functionally into another type of differentiated cell. If some non-neuronal cells in the nervous system can be reprogrammed into neurons, it would be possible to fundamentally treat diseases associated with neuronal dysfunction or neuronal death.

[0005] Recent studies have shown that regulating the expression of certain genes in the nervous system can guide the conversion of non-neuronal cells into neurons. For example, inhibiting PTBP1 can induce the transdifferentiation of glial cells into neurons. However, these studies are still in the early stages of development, and the field still lacks effective means to convert non-neuronal cells into neurons.

[0006] Summary of the Invention

[0007] One object of the present disclosure is to provide a method for generating neuronal cells from glial cells in vivo.

[0008] Another object of the present disclosure is to provide an inhibitor of the expression or activity of one or more genes or their RNA or encoded proteins selected from the following for preventing and / or treating diseases associated with neuronal dysfunction or death: RCOR1, RCOR2, RCOR3, Sin3a, Sin3b, HDAC1, HDAC2, KDM1A, PHF21A, BAF53a, G9a, USP14, HuR, BrG1, EZH2, CDYL, HMG20B.

[0009] Another object of the present disclosure is to provide an enhancer of the expression or activity of one or more genes or their RNA or encoded proteins selected from the following for preventing and / or treating diseases associated with neuronal dysfunction or death: DPYSL2, BAF45b, SCF, HuB, HuC, HuD, CYP1B1, BTRC.

[0010] In one embodiment, the present disclosure provides a method for generating neuronal cells from glial cells, comprising transdifferentiating or reprogramming the glial cells into neurons using one or more inhibitors of expression or activity of genes or their RNA or encoded proteins selected from the group consisting of RCOR1, RCOR2, RCOR3, Sin3a, Sin3b, HDAC1, HDAC2, KDM1A, PHF21A, BAF53a, G9a, USP14, HuR, BrG1, EZH2, CDYL, and HMG20B, wherein the inhibitor reduces the expression or activity of the genes or their RNA or encoded proteins, the method comprising:

[0011] (i) providing the inhibitor;

[0012] (ii) providing the glial cells;

[0013] (iii) contacting the inhibitor with the glial cells in an amount effective to cause the glial cells to transdifferentiate or reprogram into neuronal cells; and

[0014] (iv) optionally, identifying and / or isolating transdifferentiated or reprogrammed cells.

[0015] In one embodiment, the present disclosure provides a method for generating neuronal cells from glial cells, comprising transdifferentiating or reprogramming the glial cells into neurons using one or more enhancers of expression or activity of genes or their RNA or encoded proteins selected from the group consisting of DPYSL2, BAF45b, SCF, HuB, HuC, HuD, CYP1B1, and BTRC, wherein the enhancer increases the expression or activity of the genes or their RNA or encoded proteins, and the method comprises:

[0016] (i) providing the enhancer;

[0017] (ii) providing the glial cells;

[0018] (iii) contacting the enhancer with the glial cells in an amount effective to cause the glial cells to transdifferentiate or reprogram into neuronal cells; and

[0019] (iv) optionally, identifying and / or isolating transdifferentiated or reprogrammed cells.

[0020] In a preferred embodiment, the glial cells are mammalian glial cells, including glial cells of human, non-human primate, mouse, and rat species.

[0021] In a more preferred embodiment, the mammalian glial cell comprises an astrocyte, an oligodendrocyte, a microglia, an NG2 cell, a Muller glial cell, a glioma cell, or a spiral ganglion glial cell.

[0022] In a more preferred embodiment, the glial cells are astrocytes or Müller glial cells.

[0023] In a preferred embodiment, the astrocytes are derived from the cerebrum, midbrain, cerebellum, brainstem, or spinal cord.

[0024] In a more preferred embodiment, the astrocytes are derived from the striatum or substantia nigra.

[0025] In a more preferred embodiment, the Müller glial cells are derived from the retina.

[0026] In a more preferred embodiment, the spiral ganglion glial cells are derived from the inner ear or vestibule.

[0027] In a preferred embodiment, the neuronal cells are mammalian neurons, including neurons of humans, non-human primates, rats, and mice.

[0028] In a more preferred embodiment, the neuronal cells are selected from dopamine neurons, 5-HT neurons, NE neurons, ChAT neurons, motor neurons, GABA neurons, glutamatergic neurons, spinal neurons, spinal motor neurons, spinal sensory neurons, photoreceptor cells (rods and cones), bipolar cells, amacrine cells, retinal ganglion cells (RGCs), cochlear neurons, pyramidal neurons, interneurons, medium spiny neurons (MSNs), Purkinje cells, granule cells, olfactory sensory neurons, periglomerular cells or a combination thereof, more preferably dopamine neurons, retinal ganglion cells and photoreceptor cells.

[0029] In a more preferred embodiment, the glial cells are astrocytes; and the neuronal cells are dopamine neurons.

[0030] In a more preferred embodiment, the glial cells are Müller glial cells; and the neuronal cells are RGCs or photoreceptor cells.

[0031] In another embodiment, the present disclosure provides a use of one or more inhibitors of the expression or activity of genes or their RNA or encoded proteins selected from the following: RCOR1, RCOR2, RCOR3, Sin3a, Sin3b, HDAC1, HDAC2, KDM1A, PHF21A, BAF53a, G9a, USP14, HuR, BrG1, EZH2, CDYL, HMG20B in the preparation of a medicament for preventing and / or treating diseases associated with neuronal dysfunction or death, wherein the inhibitor reduces the expression or activity of the gene or its RNA or its encoded protein.

[0032] In another embodiment, the present disclosure provides a use of one or more enhancers of the expression or activity of genes or their RNA or encoded proteins selected from the following: DPYSL2, BAF45b, SCF, HuB, HuC, HuD, CYP1B1, BTRC in the preparation of a medicament for preventing and / or treating diseases associated with neuronal dysfunction or death, wherein the enhancer increases the expression or activity of the gene or its RNA or its encoded protein.

[0033] In a preferred embodiment, the medicament is formulated for in vivo administration to the nervous system, visual system, and auditory system, including the striatum, substantia nigra, ventral tegmental area of ​​the midbrain, spinal cord, hypothalamus, dorsal midbrain, cerebral cortex, hippocampus, cerebellum, subretina, vitreous cavity, inner ear cochlea, and vestibule.

[0034] In a more preferred embodiment, the medicament is formulated for administration to the striatum, substantia nigra, subretinal and vitreous cavity.

[0035] In a preferred embodiment, the disease associated with neuronal dysfunction or death is a nervous system disease, including Parkinson's disease, schizophrenia, depression, Alzheimer's disease, Huntington's disease, epilepsy, sleep disorders, ataxia, PolyQ disease, cerebral ischemia, brain injury, addiction, motor neuron disease, amyotrophic lateral sclerosis, spinal muscular atrophy, Pick's disease, visual system diseases associated with RGC or photoreceptor cell dysfunction or death, blindness, and deafness.

[0036] In a more preferred embodiment, the disease associated with neuronal dysfunction or death is Parkinson's disease and a visual system disease associated with RGC or photoreceptor cell dysfunction or death.

[0037] In another embodiment, the present disclosure provides a method for generating retinal ganglion cells (RGCs) or photoreceptor cells from Müller glial cells, comprising transdifferentiating or reprogramming the Müller glial cells into RGCs or photoreceptor cells using one or more inhibitors of the expression or activity of genes or their RNA or proteins encoded therein selected from the group consisting of RCOR1, RCOR2, RCOR3, Sin3a, Sin3b, HDAC1, HDAC2, KDM1A, PHF21A, BAF53a, G9a, USP14, HuR, BrG1, EZH2, CDYL, and HMG20B, wherein the inhibitor reduces the expression or activity of the genes or their RNA or proteins encoded therein.

[0038] In another embodiment, the present disclosure provides a method for generating retinal ganglion cells (RGCs) or photoreceptor cells from Müller glial cells, comprising transdifferentiating or reprogramming the Müller glial cells into RGCs or photoreceptor cells using one or more enhancers of the expression or activity of genes or their RNA or proteins encoded therein selected from the group consisting of DPYSL2, BAF45b, SCF, HuB, HuC, HuD, CYP1B1, and BTRC, wherein the enhancer increases the expression or activity of the genes or their RNA or proteins encoded therein.

[0039] In a preferred embodiment, the Müller glial cells are derived from the retina.

[0040] In a preferred embodiment, the photoreceptor cells include rod cells and cone cells.

[0041] In another embodiment, the present disclosure provides a use of one or more inhibitors of the expression or activity of genes or their RNA or encoded proteins selected from the following: RCOR1, RCOR2, RCOR3, Sin3a, Sin3b, HDAC1, HDAC2, KDM1A, PHF21A, BAF53a, G9a, USP14, HuR, BrG1, EZH2, CDYL, HMG20B in the preparation of a medicament, wherein the medicament is used to prevent and / or treat visual system diseases associated with loss of function or death of RGCs or photoreceptor cells, wherein the inhibitor reduces the expression or activity of the gene or its RNA or its encoded protein.

[0042] In another embodiment, the present disclosure provides a use of one or more enhancers of the expression or activity of genes or their RNA or encoded proteins selected from the following: DPYSL2, BAF45b, SCF, HuB, HuC, HuD, CYP1B1, BTRC in the preparation of a medicament for preventing and / or treating visual system diseases associated with RGC or photoreceptor cell dysfunction or death, wherein the enhancer increases the expression or activity of the gene or its RNA or its encoded protein.

[0043] In a preferred embodiment, the medicament is formulated for administration to the visual system.

[0044] In a more preferred embodiment, the drug is formulated for subretinal or vitreous administration, wherein the drug acts by acting on Müller glial cells.

[0045] In a preferred embodiment, the neurological disease associated with RGC dysfunction or death is selected from: vision impairment caused by RGC cell death, glaucoma, age-related RGC pathology, diabetic retinopathy, optic nerve damage, retinal ischemia or hemorrhage, Leber hereditary optic neuropathy, or a combination thereof.

[0046] In a preferred embodiment, the visual system disease associated with loss of photoreceptor cell function or death is selected from: photoreceptor cell degeneration or death caused by injury or degenerative lesions, macular degeneration, retinitis pigmentosa, diabetes-related blindness, night blindness, color blindness, hereditary blindness, congenital amaurosis or a combination thereof.

[0047] In a preferred embodiment, the inhibitor or agonist is selected from the group consisting of: antibodies, small molecule compounds, mRNA, microRNA, siRNA, shRNA, antisense oligonucleotides, binding proteins or protein domains, polypeptides, nucleic acid aptamers, gene editors, PROTACs, expression vectors comprising promoters, endogenous expression activators, protein analogs or enhancers, artificially synthesized or modified inhibitors or enhancers of the above, or combinations thereof.

[0048] In another embodiment, the present disclosure provides a pharmaceutical composition, a kit, or a test kit comprising one or more inhibitors of the expression or activity of genes or their RNA or proteins encoded therein selected from the group consisting of RCOR1, RCOR2, RCOR3, Sin3a, Sin3b, HDAC1, HDAC2, KDM1A, PHF21A, BAF53a, G9a, USP14, HuR, BrG1, EZH2, CDYL, HMG20B; and / or one or more enhancers of the expression or activity of genes or their RNA or proteins encoded therein selected from the group consisting of DPYSL2, BAF45b, SCF, HuB, HuC, HuD, CYP1B1, BTRC.

[0049] In a preferred embodiment, the inhibitor comprises:

[0050] (a) a gene editing protein or its expression vector, and

[0051] (b) one or more gRNAs or their expression vectors, wherein the gRNA is a DNA or RNA that guides the gene editing protein to specifically bind to the gene.

[0052] In a preferred embodiment, the editing system comprises: a CRISPR system (including a CRISPR / dCas system), a ZFN system, a TALEN system, an RNA editing system, or a combination thereof.

[0053] In a preferred embodiment, the gene editing protein is an RNA-targeting gene editing protein.

[0054] In a preferred embodiment, the gRNA is an RNA-targeting gRNA.

[0055] In a preferred embodiment, the enhancer comprises: an expression vector comprising a promoter, an endogenous expression activator, a protein analog or an enhancer.

[0056] In a preferred embodiment, the pharmaceutical composition, kit or reagent box further comprises a carrier for delivering the inhibitor.

[0057] In a preferred embodiment, the carrier is a viral vector, a liposome, a nanoparticle, an exosome, a virus-like particle, preferably AAV.

[0058] In a preferred embodiment, the RNA-targeting gene editing protein is selected from: Cas13d, Cas13e, Cas13a, Cas13b, Cas13c, Cas13f and their functional domains.

[0059] In a more preferred embodiment, the RNA-targeting gene editing protein is selected from: CasRx, Cas13e, and Cas13f.

[0060] In a more preferred embodiment, the RNA-targeting gene editing protein is CasRx.

[0061] In a preferred embodiment, the pharmaceutical composition or kit or test kit comprises only a single type of gRNA or 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 different gRNAs targeting the mRNA sequence.

[0062] In another preferred embodiment, the gRNA expression vector encodes only a single type of gRNA or 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 different gRNAs targeting the mRNA sequence.

[0063] In a preferred embodiment, the expression vector comprises:

[0064] i) a nucleotide sequence encoding the gene editing protein, which is operably linked to a promoter that causes expression of the gene editing protein, and

[0065] ii) at least one nucleotide sequence encoding a gRNA targeting the mRNA or DNA sequence, wherein the nucleotide sequence is operably linked to a promoter, such as a U6 promoter, that causes the gRNA to be expressed in mammalian cells.

[0066] In a preferred embodiment, the promoter is a broad-spectrum promoter or a specific promoter.

[0067] In a more preferred embodiment, the broad-spectrum promoter is selected from CMV, CBH, CAG, PGK, SV40, EF1A, EFS, and pGlobin promoters.

[0068] In a more preferred embodiment, the specific promoter is a glial cell-specific promoter or a Muller glial (MG) cell-specific promoter.

[0069] In a more preferred embodiment, the glial cell-specific promoter is selected from the group consisting of GFAP promoter, ALDH1L1 promoter, EAAT1 / GLAST promoter, glutamine synthetase promoter, S100β promoter and EAAT2 / GLT-1 promoter, NG2 promoter, CD68 promoter, F4 / 80 promoter, or the MG cell-specific promoter is selected from the group consisting of GFAP promoter, ALDH1L1 promoter, Glast (also known as Slc1a3) promoter and Rlbp1 promoter.

[0070] In a preferred embodiment, the expression vector is contained in a viral particle.

[0071] In a preferred embodiment, the expression vector is a gene therapy vector.

[0072] In a more preferred embodiment, the gene therapy vector is a viral gene therapy vector.

[0073] In a more preferred embodiment, the expression vector is a viral vector selected from the group consisting of an adeno-associated virus (AAV) vector, a recombinant adeno-associated virus vector (rAAV), a self-complementary adeno-associated virus vector (scAAV), an adenoviral vector, a lentiviral vector, a retroviral vector, a herpes virus vector, an SV40 vector, a poxvirus vector, and combinations thereof.

[0074] In a more preferred embodiment, the expression vector is an AAV vector or rAAV.

[0075] In a preferred embodiment, the composition is topically administered to at least one of: i) glial cells in the retina; ii) glial cells in the striatum, preferably glial cells in the putamen; iii) glial cells in the substantia nigra; iv) glial cells in the inner ear; v) glial cells in the spinal cord; vi) glial cells in the prefrontal cortex; vii) glial cells in the motor cortex; viiii) glial cells in the thalamus; ix) glial cells in the ventral tegmental area (VTA); x) glial cells in the hippocampus; xi) glial cells in the cerebellum; and xii) glial cells in the brainstem.

[0076] In a preferred embodiment, the composition further comprises i) one or more neuron-related factors, or ii) at least one expression vector for expressing one or more neuron-related factors in the glial cells.

[0077] In a more preferred embodiment, the one or more neuron-related factors are selected from: AscL1, Mytl1, Ngn1, Ngn2, NeuroD1, NeuroD2, NeuroG1, Pax6, Ptbp1, P53, Zic1, Ctdsp1, miR-9, miR-9-9*, miR-124, miR-124-124*, Let-7, Let-7b, miR-132, NeuroG2, Brn2, NeuroD4, Insm1, Prox1, FoxG1, Lhx6, Bcl2, Dlx1, Dlx2, Tlx3, Gata2, Gata3, Sox11, Lhx3, IsL1, etc.

[0078] In a preferred embodiment, the pharmaceutical composition, drug kit or test kit further comprises i) one or more dopamine neuron-related factors, or ii) at least one expression vector for expressing one or more dopamine neuron-related factors in the glial cells.

[0079] In a preferred embodiment, the composition is also formulated for cell transfection, cell infection, cell endocytosis, injection, intracranial administration, spinal administration, intraocular administration, intraaural administration, inhalation, parenteral administration, intravenous administration, intramuscular administration, intradermal administration, topical administration or oral administration, as well as inducing transdifferentiation or reprogramming in vitro and transplanting the transdifferentiated or reprogrammed cells back into the body.

[0080] In a preferred embodiment, the AAV vector comprises:

[0081] i) a nucleotide sequence encoding the gene editing protein, which is operably linked to a promoter that causes the gene editing protein to be expressed in glial cells; and

[0082] ii) at least one nucleotide sequence encoding a gRNA targeting the mRNA or DNA sequence, which is operably linked to a promoter, such as a U6 promoter, that causes the gRNA to be expressed in mammalian cells.

[0083] In a preferred embodiment, the promoter is a broad-spectrum promoter or a specific promoter.

[0084] In a more preferred embodiment, the broad-spectrum promoter is selected from CMV, CBH, CAG, PGK, SV40, EF1A, EFS, and pGlobin promoters.

[0085] In a more preferred embodiment, the specific promoter is a glial cell-specific promoter or a Muller glial (MG) cell-specific promoter.

[0086] In a more preferred embodiment, the glial cell-specific promoter is selected from the group consisting of GFAP promoter, ALDH1L1 promoter, EAAT1 / GLAST promoter, glutamine synthetase promoter, S100β promoter, EAAT2 / GLT-1 promoter, NG2 promoter, CD68 promoter, and F4 / 80 promoter.

[0087] In a more preferred embodiment, the MG cell-specific promoter is selected from the group consisting of GFAP promoter, ALDH1L1 promoter, Glast (also known as Slc1a3) promoter, and Rlbp1 promoter.

[0088] In a preferred embodiment, the transdifferentiation efficiency of glial cells is at least 0.1%, or at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 6 8%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, or more.

[0089] In another preferred embodiment, the disease associated with neuronal dysfunction or death is selected from: Parkinson's disease, schizophrenia, depression, vision impairment caused by RGC cell death, diabetic retinopathy, glaucoma, age-related RGC lesions, optic nerve damage, retinal ischemia or hemorrhage, Leber hereditary optic neuropathy, photoreceptor cell degeneration or death caused by damage or degenerative lesions, macular degeneration, retinitis pigmentosa, diabetes-related blindness, night blindness, color blindness, hereditary blindness, congenital amaurosis or a combination thereof.

[0090] In another preferred embodiment, the RGCs can be integrated into the visual pathway and improve visual function.

[0091] In another preferred embodiment, the RGCs can achieve functional projections to the central visual area and improve visual function.

[0092] In another preferred embodiment, the improving visual function is improving the visual function of a mammal suffering from a retinal disease caused by neurodegeneration.

[0093] In another preferred embodiment, the MG cells are transdifferentiated into RGC cells and also differentiated into axonal cells.

[0094] In another preferred embodiment, a plurality of glial cells in the striatum are reprogrammed or transdifferentiated, and wherein at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29% or at least 30% of the glial cells are converted into dopamine neurons.

[0095] In another preferred embodiment, the mammal includes a mammal suffering from a disease associated with neuronal loss of function or death.

[0096] In another preferred embodiment, the mammal comprises a human or a non-human mammal.

[0097] In another preferred embodiment, the non-human mammal includes a rodent (such as a mouse, rat, or rabbit) or a primate (such as a monkey).

[0098] In another preferred embodiment, the gene editor is expressed by a glial cell-specific promoter (e.g., the GFAP promoter).

[0099] In another preferred embodiment, the gene editor comprises one or more gRNAs and a gene editing protein.

[0100] In another preferred embodiment, the gRNA guides the gene editing protein to specifically bind to the RNA of the gene.

[0101] In another preferred embodiment, the gRNA guides the gene editing protein to specifically bind to the mRNA of the gene.

[0102] In another preferred embodiment, the nucleotide sequence of the gRNA is shown in Table 1 below.

[0103] Table 1: Nucleotide sequences of gRNA and target sequences

[0104] As used herein, the letter "u" is used interchangeably with the letter "t" in the context of RNA sequences to represent uridine or uridylic acid.

[0105] In another preferred embodiment, the source of the gene editing protein is selected from: Streptococcus pyogenes, Staphylococcus aureus, Acidaminococcus sp, Lachnospiraceae bacterium, Ruminococcus flavefaciens, or a combination thereof.

[0106] In another preferred embodiment, the gene or its RNA or its encoded protein is derived from a mammal; preferably, from a human, monkey, mouse, rat, or rabbit; more preferably, from a human.

[0107] In another preferred embodiment, the gene includes a wild-type gene and a mutant gene.

[0108] In another preferred embodiment, the mutant type includes a mutant form in which the function of the encoded protein is not changed after the mutation (ie, the function is the same or substantially the same as that of the wild-type encoded protein).

[0109] In another preferred embodiment, the polypeptide encoded by the mutant gene is identical or substantially identical to the polypeptide encoded by the wild-type gene.

[0110] In another preferred embodiment, the mutant gene comprises a polynucleotide having a homology ≥80% (preferably ≥90%, ≥91%, ≥92%, ≥93% or ≥94%, more preferably ≥95%, ≥96% or ≥97%, more preferably ≥98% or 99%) compared to the wild-type gene.

[0111] In another preferred embodiment, the mutant gene comprises a polynucleotide having 1-60 (preferably 1-30, more preferably 1-10) nucleotides truncated or added to the 5' end and / or 3' end of the wild-type gene.

[0112] In another preferred embodiment, the gene comprises a cDNA sequence, a genomic sequence, or a combination thereof.

[0113] In another preferred embodiment, the protein includes an active fragment or a derivative thereof.

[0114] In another preferred embodiment, the homology of the active fragment or its derivative to the gene or its RNA or its encoded protein is at least 90%, 91%, 92%, 93% or 94%, preferably 95%, 96% or 97%, more preferably 98%, 99%.

[0115] In another preferred embodiment, the active fragment or derivative thereof has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% activity.

[0116] In another preferred embodiment, the amino acid sequence of the protein is selected from the group consisting of:

[0117] (i) a polypeptide having an amino acid sequence as shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, or 49;

[0118] (ii) a polypeptide derived from (i) having the function of the protein, formed by substituting, deleting or adding one or several (e.g., 1-10) amino acid residues of the amino acid sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49; or

[0119] (iii) an amino acid sequence that is ≥90%, ≥91%, ≥92%, ≥93% or ≥94% (preferably ≥95%, ≥96% or ≥97%, more preferably ≥98% or 99%) identical to the amino acid sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, and has the function of the protein.

[0120] In another preferred embodiment, the nucleotide sequence of the gene is selected from:

[0121] (a) a polynucleotide encoding a polypeptide having the amino acid sequence shown above;

[0122] (b) polynucleotides of the sequences shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50;

[0123] (c) a polynucleotide having a nucleotide sequence that is ≥90%, ≥91%, ≥92%, ≥93% or ≥94% (preferably ≥95%, ≥96% or ≥97%, more preferably ≥98% or 99%) identical to the nucleotide sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50;

[0124] (d) a polynucleotide having a nucleotide sequence as shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, truncated at the 5' end and / or 3' end or added with 1-60 (preferably 1-30, more preferably 1-10) nucleotides;

[0125] (e) A polynucleotide complementary to the polynucleotide of any one of (a) to (d).

[0126] In another preferred embodiment, the protein is as shown in the above amino acid sequence.

[0127] In another preferred embodiment, the nucleic acid encoding the protein is as shown in the above nucleotide sequence.

[0128] In another preferred embodiment, the regions targeted by the inhibitor (such as a gene editing protein) are positions 121931-121963 (RCOR1), positions 2364-2393 (RCOR2), positions 53556-53585 (RCOR3), positions 59338-59367 (Sin3a), positions 12378-12406 (Sin3b), positions 24648-24678 (HDAC1) and positions 60126-60154 (KDM1A) of the gene sequence.

[0129] In another preferred embodiment, the inhibitor or enhancer inhibits or enhances the activity and / or expression level of the gene or its RNA or its encoded protein.

[0130] In another preferred embodiment, the concentration of the inhibitor or enhancer (virus titer) is > 1×10 12 .

[0131] In another preferred embodiment, the inhibitory or enhancing agent has an inhibition rate or enhancement rate of greater than 90% on the activity and / or expression of the gene, its RNA or its encoded protein, and / or the like, preferably 90%-95%.

[0132] In another preferred embodiment, the inhibitor or enhancer targets astrocytes of brain tissue.

[0133] In another preferred embodiment, the inhibitor or enhancer targets MG cells of the retina.

[0134] In another preferred embodiment, the gRNA guides the gene editing protein to specifically bind to the mRNA of the gene.

[0135] In another preferred embodiment, the composition comprises a pharmaceutical composition.

[0136] In another preferred embodiment, the composition further comprises other drugs for preventing and / or treating diseases associated with neuronal function loss or death.

[0137] In another preferred embodiment, the composition further comprises other drugs for treating neurological diseases associated with neuronal death.

[0138] In another preferred embodiment, the composition further comprises other drugs for preventing and / or treating retinal diseases.

[0139] In another preferred embodiment, the expression vector of the gene editing protein includes a vector targeting glial cells.

[0140] In another preferred embodiment, the expression vector of the gene editing protein includes a vector targeting astrocytes in brain tissue.

[0141] In another preferred embodiment, the expression vector of the gene editing protein includes a vector targeting retinal MG cells.

[0142] In another preferred embodiment, the vector comprises AAV2 or AAV9.

[0143] In another preferred embodiment, the gene encoding the gene editing protein and the gRNA are located in the same expression vector (such as an AAV vector).

[0144] In another preferred embodiment, the expression vector of the gene editing protein and the expression vector of the gRNA are the same expression vector (such as an AAV vector).

[0145] In another preferred embodiment, the expression vector further comprises a glial cell-specific promoter (eg, GFAP promoter) for driving the expression of the gene editing protein.

[0146] In another preferred embodiment, the dosage form of the composition is selected from: a lyophilized preparation, a liquid preparation, or a combination thereof.

[0147] In another preferred embodiment, the composition is in the form of a liquid preparation.

[0148] In another preferred embodiment, the composition is in the form of an injection.

[0149] In another preferred embodiment, other drugs for preventing and / or treating diseases associated with neuronal dysfunction or death are selected from: dopamine prodrugs, non-ergot dopamine receptor agonists, monoamine oxidase B inhibitors, or combinations thereof.

[0150] In another preferred embodiment, the composition is a cell preparation.

[0151] In another preferred embodiment, the expression vector of the gene editing protein and the expression vector of the gRNA are the same vector or different vectors.

[0152] In another preferred embodiment, the weight ratio of component (a) to component (b) is 100:1-0.01:1, preferably, 10:1-0.1:1, more preferably, 2:1-0.5:1.

[0153] In another preferred embodiment, in the composition, the content of component (a) is 0.001%-99%, preferably, 0.1%-90%, more preferably, 1%-70%.

[0154] In another preferred embodiment, in the composition, the content of component (b) is 0.001%-99%, preferably, 0.1%-90%, more preferably, 1%-70%.

[0155] In another preferred embodiment, in the composition, the content of component (c) is 1%-99%, preferably, 10%-90%, more preferably, 30%-70%.

[0156] In another preferred embodiment, in the composition, the component (a), component (b) and optional component (c) account for 0.01-99.99 wt%, preferably 0.1-90 wt%, more preferably 1-80 wt% of the total weight of the composition.

[0157] In another embodiment, the present disclosure provides a kit comprising:

[0158] (a1) a first container, and a gene editing protein or its expression vector, or a drug containing a gene editing protein or its expression vector, located in the first container, wherein the gene editing protein is selected from the group consisting of Cas13d, Cas13e, Cas13a, Cas13b, Cas13c, Cas13f, an RNA-targeted gene editing protein, or a combination thereof;

[0159] (b1) a second container, and a gRNA or its expression vector, or a drug containing the gRNA or its expression vector, located in the second container, wherein the gRNA guides the gene editing protein to specifically bind to the DNA or RNA of the gene.

[0160] In another preferred embodiment, the kit further comprises:

[0161] (c1) a third container, and other drugs for preventing and / or treating diseases associated with neuronal dysfunction or death, and / or containing other drugs for preventing and / or treating retinal diseases, and / or containing other drugs for treating neurological diseases associated with neuronal death, located in the third container.

[0162] In another preferred embodiment, the first container, the second container and the third container are the same or different containers.

[0163] In another preferred embodiment, the drug in the first container is a single-ingredient preparation containing a gene editing protein or an expression vector thereof.

[0164] In another preferred embodiment, the drug in the second container is a single-ingredient preparation containing gRNA or its expression vector.

[0165] In another preferred embodiment, the drug in the third container is a single-ingredient preparation containing other drugs for treating neurological diseases associated with neuronal death.

[0166] In another preferred embodiment, the dosage form of the drug is selected from: a lyophilized preparation, a liquid preparation, or a combination thereof.

[0167] In another preferred embodiment, the dosage form of the drug is an oral dosage form or an injection dosage form. In another preferred embodiment, the kit further comprises instructions.

[0168] Specifically, this application also involves the following solutions:

[0169] Item 1. A method for transdifferentiating non-neuronal cells of a mammal into neurons, comprising:

[0170] Provided are negative regulators capable of reducing the expression of negative regulatory genes, including RCOR1, RCOR2, RCOR3, Sin3a, Sin3b, HDAC1, HDAC2, KDM1A, PHF21A, BAF53a, G9a, USP14, HuR, BrG1, EZH2, CDYL, or HMG20B, and / or

[0171] Providing positive regulators capable of increasing the expression of positively regulated genes, wherein the positively regulated genes include DPYSL2, BAF45b, SCF, HuB, HuC, HuD, CYP1B1, or BTRC,

[0172] An effective amount of the negative regulator or the positive regulator contacts the non-neuronal cells to induce the non-neuronal animal cells to transdifferentiate into neurons.

[0173] In one embodiment, the method is an in vitro, ex vivo or in vivo method.

[0174] In a specific embodiment, the sequences of the proteins involved in the negative regulation of genes are as follows: RCOR1 is the amino acid sequence set forth in SEQ ID NO: 1. RCOR2 is the amino acid sequence set forth in SEQ ID NO: 3. RCOR3 is the amino acid sequence set forth in SEQ ID NO: 5. Sin3a is the amino acid sequence set forth in SEQ ID NO: 7. Sin3b is the amino acid sequence set forth in SEQ ID NO: 9. HDAC1 is the amino acid sequence set forth in SEQ ID NO: 11. HDAC2 is the amino acid sequence set forth in SEQ ID NO: 13. KDM1A is the amino acid sequence set forth in SEQ ID NO: 15. PHF21A is the amino acid sequence set forth in SEQ ID NO: 17. BAF53a is the amino acid sequence set forth in SEQ ID NO: 19. G9a is the amino acid sequence set forth in SEQ ID NO: 21. USP14 is the amino acid sequence set forth in SEQ ID NO: 23. HuR is the amino acid sequence set forth in SEQ ID NO: 25. BRG1 is the amino acid sequence set forth in SEQ ID NO: 27. EZH2 is the amino acid sequence set forth in SEQ ID NO: 29. CDYL is the amino acid sequence set forth in SEQ ID NO: 31. HMG20B has the amino acid sequence shown in SEQ ID NO: 33.

[0175] In one specific embodiment, the sequences of the proteins involved in the positive regulation of genes are as follows: DPYSL2 has the amino acid sequence set forth in SEQ ID NO:35. BAF45b has the amino acid sequence set forth in SEQ ID NO:37. SCF has the amino acid sequence set forth in SEQ ID NO:39. HuB has the amino acid sequence set forth in SEQ ID NO:41. HuC has the amino acid sequence set forth in SEQ ID NO:43. HuD has the amino acid sequence set forth in SEQ ID NO:45. CYP1B1 has the amino acid sequence set forth in SEQ ID NO:47. BTRC has the amino acid sequence set forth in SEQ ID NO:49.

[0176] Item 2. The method according to Item 1, wherein the non-neuronal cells are derived from humans.

[0177] Item 3. The method of Item 1 or Item 2, wherein the non-neuronal cells are stem cells, progenitor cells or terminally differentiated cells; preferably, glial cells; more preferably, the glial cells are astrocytes, oligodendrocytes, microglia, NG2 cells, Müller glial cells, glioma cells or spiral ganglion glial cells; more preferably, the glial cells are astrocytes or Müller glial cells.

[0178] Item 4. The method of Item 3, wherein the non-neuronal cells are derived from the brain. Preferably, the non-neuronal cells are derived from the cerebrum, midbrain, cerebellum, brainstem, or spinal cord; more preferably, they are derived from the striatum or substantia nigra in the brain.

[0179] Item 5. The method according to Item 4, wherein the non-neuronal cells are astrocytes derived from the brain, preferably astrocytes derived from the striatum or substantia nigra.

[0180] Item 6. The method according to Item 4, wherein the non-neuronal cells are derived from the eye, and preferably, the non-neuronal cells are Müller glial cells from the eye.

[0181] Item 7. The method of any one of Items 1 to 6, wherein the neuron is a dopaminergic neuron, a retinal ganglion cell, a photoreceptor cell, a 5-HT neuron, a NE neuron, a ChAT neuron, a motor neuron, a GABA neuron, a glutamatergic neuron, a spinal cord neuron, a spinal cord motor neuron, a spinal cord sensory neuron, a bipolar cell, an amacrine cell, a cochlear neuron, a pyramidal neuron, an interneuron, a medium spiny neuron, a Purkinje cell, a granule cell, an olfactory sensory neuron, or a periglomerular cell, or a combination thereof.

[0182] Item 8. The method according to any one of Items 1 to 7, wherein the negative regulatory gene is RCOR1, RCOR2, RCOR3, Sin3a, Sin3b, KDM1A, BAF53a, G9a, HuR, BrG1, or EZH2, and the non-neuronal animal cells are derived from the brain or eyes.

[0183] Item 9. The method of Item 8, wherein the negative regulatory gene is RCOR1, RCOR2, RCOR3 or G9a, and the non-neuronal animal cells are derived from the brain; preferably, the non-neuronal cells are astrocytes from the brain; more preferably, the non-neuronal cells are astrocytes from the striatum or substantia nigra.

[0184] Item 10. The method of Item 8, wherein the negative regulatory gene is Sin3a, Sin3b, KDM1A, BAF53a, G9a, HuR, BrG1, CDYL, or EZH2, and the non-neuronal cells are derived from the eye; preferably, the non-neuronal cells are Muller glial cells from the eye.

[0185] Item 11. The method of Item 10, wherein the negative regulatory gene is HuR and the neurons are retinal ganglion cells.

[0186] Item 12. The method of Item 10, wherein the negative regulatory gene is Sin3a, KDM1A, BAF53a, G9a, HuR, BrG1, CDYL, or EZH2, and the neuron is a photoreceptor cell.

[0187] Item 13. The method of any one of Items 1 to 7, wherein the positively regulated gene is DPYSL2, BAF45b, SCF, HuB, HuC, HuD, CYP1B1, or BTRC, and the non-neuronal cells are from the brain; preferably, the non-neuronal cells are from the striatum, substantia nigra, ventral tegmental area of ​​the midbrain, spinal cord, hypothalamus, dorsal midbrain, cerebral cortex, hippocampus, and cerebellum; more preferably, they are from the striatum.

[0188] Item 14. The method according to Item 13, wherein the positive regulatory gene is SCF or HuB.

[0189] Item 15. The method of Item 13, wherein the positive regulatory gene is HuB or BTRC, and an effective amount of the positive regulator contacts the non-neuronal cells to induce the transdifferentiation of the non-neuronal cells into dopaminergic neurons.

[0190] Item 16. The method according to any one of Items 13 to 15, wherein the non-neuronal cells are astrocytes; more preferably, the non-neuronal cells are astrocytes from the striatum or substantia nigra.

[0191] Item 17. The method according to any one of Items 1 to 7, wherein the positively regulated gene is DPYSL2, BAF45b, SCF, HuC, HuD, or CYP1B1, and the non-neuronal cells are from the eye; preferably, the non-neuronal cells are Muller glial cells from the eye.

[0192] Item 18. The method according to Item 17, wherein the positive regulatory gene is SCF, HuD, or CYP1B1, and the neuron is a retinal ganglion cell.

[0193] Item 19. The method of Item 17, wherein the positively regulated gene is DPYSL2 or BAF45b, and the neurons are photoreceptor cells.

[0194] Item 20. The method according to any one of Items 1 to 19, wherein the reducing the expression of the negative regulatory gene is capable of reducing the gene level of the negative regulatory gene, or reducing the mRNA level of the negative regulatory gene, or reducing the expression level of the protein encoded by the negative regulatory gene;

[0195] The improvement of the positive regulatory gene expression is to increase the gene level of the positive regulatory gene, or to increase the mRNA level of the positive regulatory gene, or to increase the expression level of the protein encoded by the positive regulatory gene.

[0196] Item 21. The method of Item 20, wherein the negative regulator is selected from a gene editing tool or an epigenetic regulation tool that reduces the expression of a negative regulatory gene; an inhibitor of a negative regulatory gene, an inhibitor of the activity of a negative regulatory gene, or an activator of degradation of the protein encoded by the negative regulatory gene.

[0197] Item 22. The method of Item 21, wherein the inhibitor is: an inhibitory antibody of a negative regulatory gene; or a small molecule inhibitor of a negative regulatory gene; or an inhibitory mRNA, microRNA, siRNA, shRNA, antisense oligonucleotide, binding protein or protein domain, polypeptide, nucleic acid aptamer, or PROTAC of a negative regulatory gene; or an inhibitory binding protein or ligand of a negative regulatory gene.

[0198] Item 23. The method of Item 21, wherein the negative regulator contains the gRNA and gene editing protein as described in any one of SEQ ID NOs: 51-67.

[0199] Item 24. The method of Item 20, wherein the positive regulator is selected from an epigenetic regulatory tool capable of increasing the expression of a positive regulatory gene, an activator of positive regulatory gene expression, an inhibitor of degradation of a protein encoded by a positive regulatory gene, a stabilizer of positive regulatory gene mRNA, or an exogenous positive regulatory gene or a functional fragment of a positive regulatory gene.

[0200] Item 25. The method of Item 24, wherein the activator is: an agonistic antibody of a positively regulated gene; or a small molecule agonist of a positively regulated gene; or an agonistic binding protein or ligand of a positively regulated gene; or an inhibitor of a competitive gene of a positively regulated gene.

[0201] Item 26. A method as described in Item 24, wherein the positive regulator contains a nucleic acid sequence as shown in SEQ ID NO: 36, 38, 40, 42, 44, 46, 48, or 50, or a functional fragment of a nucleic acid sequence as shown in SEQ ID NO: 36, 38, 40, 42, 44, 46, 48, or 50.

[0202] Item 27. The gene editing tool of Item 21, wherein the gene editing tool comprises a gene editing system or an expression vector thereof, and the gene editing system is selected from: a CRISPR system (including a CRISPR / Cas system), a ZFN system, a TALEN system, or a combination thereof.

[0203] Item 28. The method according to Item 27, wherein the CRISPR system is used to reduce the expression or activity of negative regulatory genes; preferably, the CRISPR system contains a nucleic acid encoding a Cas enzyme or a functional domain of a Cas enzyme and a gRNA targeting the cell transdifferentiation factor; more preferably, the Cas enzyme is Cas13; more preferably, the Cas enzyme is Cas13d, Cas13X, Cas13a, Cas13b, Cas13c, or Cas13Y; more preferably, the Cas enzyme is CasRx.

[0204] Item 29. The method according to Item 20, wherein the negative regulator or positive regulator is carried by a carrier; preferably, the carrier is a viral vector, lipid nanoparticles (LNP), liposomes, cationic polymers (such as PEI), nanoparticles, exosomes, or virus-like particles; more preferably, the carrier is an AAV vector or lipid nanoparticles.

[0205] Item 30. The method according to any one of Items 1 to 29, wherein the effective amount of the negative regulator or the positive regulator is contacted with the non-neuronal cells in vitro to induce the non-neuronal cells to transdifferentiate into neurons in vitro; or

[0206] The effective amount of the negative regulator or the positive regulator contacts the non-neuronal cells in vivo to induce the non-neuronal cells to transdifferentiate into neurons in vitro.

[0207] Item 31. Use of the negative regulator or positive regulator according to any one of Items 1 to 30 for the preparation of a drug for preventing or treating diseases associated with neuronal damage or neuronal death.

[0208] Item 32. Use of the negative regulator or positive regulator according to any one of Items 1 to 30 for preventing or treating diseases associated with neuronal damage or neuronal death.

[0209] Item 33. The use as described in Item 31 or 32, wherein the drug is formulated as an agent for in vivo administration to the nervous system, visual system and auditory system, for example, in vivo administration to the striatum, substantia nigra, ventral tegmental area of ​​the midbrain, spinal cord, hypothalamus, dorsal midbrain, cerebral cortex, hippocampus, cerebellum, subretinal space, vitreous cavity, inner ear cochlea and vestibule, preferably striatum, subretinal space and vitreous cavity.

[0210] Item 34. The use as described in Item 31 or 32, wherein the disease associated with neuronal damage or neuronal death is selected from Parkinson's disease, visual system diseases associated with loss of function or death of RGC or photoreceptor cells, Alzheimer's disease, brain injury, Huntington's disease, epilepsy, depression, sleep disorders, cerebral ischemia, motor neuron disease, amyotrophic lateral sclerosis, spinal muscular atrophy, ataxia, PolyQ disease, schizophrenia, addiction, Pick's disease, blindness, and deafness; preferably, it is a visual system disease associated with Parkinson's disease and loss of function or death of RGC or photoreceptor cells.

[0211] Item 35. The use as described in Item 34, wherein the visual system disease associated with loss of RGC or photoreceptor cell function or death is preferably selected from: vision impairment caused by death of RGC cells or photoreceptor cells, glaucoma, age-related RGC lesions, optic nerve damage, age-related macular degeneration (AMD), diabetic retinopathy, retinal ischemia or hemorrhage, Leber hereditary optic neuropathy, or a combination thereof; the visual system disease associated with loss of photoreceptor cell function or death is preferably selected from: photoreceptor cell degeneration or death caused by damage or degenerative lesions, macular degeneration, retinitis pigmentosa, diabetes-related blindness, night blindness, color blindness, hereditary blindness, congenital amaurosis, or a combination thereof.

[0212] Item 36. The use according to Item 31 or 32, wherein the neurons are dopaminergic neurons, 5-HT neurons, NE neurons, ChAT neurons, GABA neurons, glutamatergic neurons, motor neurons, photoreceptor cells (such as rod cells and cone cells), retinal ganglion cells (RGCs), cochlear nerve cells (such as cochlear spiral ganglion cells and vestibular neurons), or medium spiny neurons (MSNs) or a combination thereof, preferably dopaminergic neurons, retinal ganglion cells and photoreceptor cells.

[0213] Item 37. A method for preventing or treating a disease associated with neuronal damage or neuronal death, comprising administering to a subject in need thereof an effective amount of a negative regulator or a positive regulator according to any one of Items 1 to 30.

[0214] Item 38. The method according to Item 37, wherein the disease associated with neuronal damage or neuronal death is selected from Parkinson's disease, visual system diseases associated with loss of function or death of RGC or photoreceptor cells, Alzheimer's disease, brain injury, Huntington's disease, epilepsy, depression, sleep disorders, cerebral ischemia, motor neuron disease, amyotrophic lateral sclerosis, spinal muscular atrophy, ataxia, PolyQ disease, schizophrenia, addiction, Pick's disease, blindness, and deafness; preferably, it is a visual system disease associated with Parkinson's disease and loss of function or death of RGC or photoreceptor cells.

[0215] Item 39. A method as described in Item 38, wherein the visual system disease associated with RGC dysfunction or death is preferably selected from: vision impairment caused by RGC cell death, glaucoma, age-related RGC lesions, optic nerve damage, age-related macular degeneration (AMD), diabetic retinopathy, retinal ischemia or hemorrhage, Leber hereditary optic neuropathy, or a combination thereof; the visual system disease associated with photoreceptor cell dysfunction or death is preferably selected from: photoreceptor cell degeneration or death caused by injury or degenerative lesions, macular degeneration, retinitis pigmentosa, diabetes-related blindness, night blindness, color blindness, hereditary blindness, congenital amaurosis, or a combination thereof.

[0216] Item 40. A method according to Item 37, wherein the neurons are dopaminergic neurons, 5-HT neurons, NE neurons, ChAT neurons, GABA neurons, glutamatergic neurons, motor neurons, photoreceptor cells (such as rod cells and cone cells), retinal ganglion cells (RGCs), cochlear nerve cells (such as cochlear spiral ganglion cells and vestibular neurons), or medium spiny neurons (MSNs) or a combination thereof, preferably dopaminergic neurons, retinal ganglion cells and photoreceptor cells.

[0217] The technical solutions involved in this application also include:

[0218] Use of the negative or positive regulator of KDM1A or EZH2 as described in any one of Items 1 to 30 for the preparation of a medicament for preventing or treating a disease associated with optic neuron damage or optic neuron death.

[0219] In some embodiments, the disease associated with optic neuron damage or optic neuron death can be retinitis pigmentosa (RP).

[0220] It should be understood that within the scope of the present disclosure, the above-mentioned technical features of the present disclosure and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0221] Figure 1: CasRx-specific knockdown of target genes in vitro. In the N2A cell line, expression of CasRx and gRNAs targeting RCOR1, Sin3a, HDAC2, PHF21A, BAF53a, KDM1A, G9a, USP14, HuR, BrG1, EZH2, CDYL, and HMG20B efficiently knocked down RCOR1, Sin3a, HDAC2, KDM1A, PHF21A, BAF53a, G9a, USP14, HuR, BrG1, EZH2, CDYL, and HMG20B.

[0222] Figure 2: Schematic diagram of transdifferentiation of mouse astrocytes into neurons by knocking down or overexpressing different target genes. Vector 1 (AAV-GFAP-mCherry) in Figure 2A drives the expression of fluorescent protein mCherry by the astrocyte-specific promoter GFAP, vector 2 (AAV-GFAP-CasRx) drives the expression of RNA editing protein CasRx by the astrocyte-specific promoter GFAP, and vector 3 (AAV-GFAP-CasRx-sg) encodes CasRx and gRNA targeting RCOR1, Sin3a, HDAC2, KDM1A, PHF21A, BAF53a, G9a, USP14, HuR, BrG1, EZH2, CDYL, or HMG20B; Vector 1 in Figure 2B is a schematic diagram of the vector for GFAP-driven mCherry, and vector 4 is a schematic diagram of the vector for GFAP-driven expression of target genes, and the target genes are DPYSL2, BAF45b, SCF, HuB, HuC, HuD, CYP1B1, or BTRC.

[0223] Figure 3: Knockdown of different negative regulatory genes in the striatum of mice resulted in the conversion of astrocytes into neurons. Figure 3A shows that the striatum of mice injected with control AAV (AAV-GFAP-mCherry + AAV-GFAP- After CasRx, AAV-GFAP-mCherry can specifically label astrocytes and will not transdifferentiate into neurons. Yellow arrows (the arrows in Figure 3A are yellow arrows) indicate astrocytes labeled with GFAP-mCherry, which are not co-labeled with NeuN, a neuron-specific marker; Figures 3B to 3M respectively show the results of astrocyte transdifferentiation into neurons by knocking down RCOR1, HDAC2, PHF21A, BAF53a, G9a, USP14, HuR, BrG1, EZH2, CDYL, HMG20B, and KDM1A in the mouse striatum using CasRx. The red signals are cells labeled with AAV-GFAP-mCherry (the leftmost column of Figures 3A to 3M is the result of mCherry labeling, and the bright parts in the figures are red). NeuN is a neuron-specific marker, and white arrows indicate cells co-labeled with red mCherry signals and NeuN (the arrows in Figures 3B to 3M are white arrows). The scale bar is 50 μm.

[0224] Figure 4: Statistical graph of the conversion of astrocytes into neurons after knockdown of different negative regulatory genes.

[0225] Figure 5: Overexpression of different positive regulatory genes converts astrocytes into neurons in the mouse striatum. Figure 5A shows a control group of AAVs (AAV-GFAP-mCherry) injected into the striatum of the mouse brain to specifically label astrocytes. Yellow arrows (the arrows in Figure 5A are yellow arrows) indicate that astrocytes labeled with GFAP-mCherry are not co-labeled with NeuN, a neuron-specific marker. Figures 5B to 5I show the results of injecting a test group of AAVs (AAV-GFAP-mCherry + AAV-GFAP-positive regulatory genes) into the striatum of the mouse brain. The positive regulatory genes are DPYSL2, BAF45b, SCF, HuB, HuC, HuD, CYP1B1, and BTRC, respectively. White arrows indicate cells co-labeled with red mCherry signals and NeuN (the leftmost column of Figures 5A to 5I shows the results of mCherry labeling; the bright areas in the figures are red, and the arrows in Figures 5B to 5I are white arrows). NeuN is a neuron-specific marker. The scale bar is 50 microns.

[0226] Figure 6: Statistics of astrocyte-to-neuron conversion after upregulation of different positively regulated genes.

[0227] Figure 7: Conversion of astrocytes into dopaminergic neurons after overexpression of BTRC or HuB in the mouse striatum. Figure 7A shows the results of injection of a control AAV (AAV-GFAP-mCherry) into the mouse striatum. Yellow arrows (the arrows in Figure 7A are yellow arrows) indicate mCherry-positive cells that do not co-label with TH, a dopaminergic neuron-specific protein marker, or NeuN, a neuron-specific marker. Figures 7B and 7C show the results of injection of a test AAV (AAV-GFAP-mCherry + AAV-GFAP-positive regulatory gene) into the mouse striatum, respectively. The positive regulatory genes are HuB and BTRC. White arrows (the arrows in Figures 7B and C are white arrows) indicate cells where the red mCherry signal co-labels with NeuN and TH. Scale bar is 50 μm. The bright part in the mCherry column is red, the bright part in the TH column is green, the bright part in the NeuN column is white, and the bright part in the DAPI column is blue.

[0228] Figure 8: Schematic diagram of overexpressing or knocking down the target gene in the retina to convert Müller glia into retinal ganglion cells or photoreceptors. Vector 1 in Figure 8A is a schematic diagram of the AAV-GFAP-EGFP-2A-Cre plasmid, in which Cre expression is driven by the astrocyte-specific promoter GFAP. Vector 2 is a schematic diagram of the AAV-GFAP-CasRx plasmid, in which the RNA editing protein CasRx is driven by the astrocyte-specific promoter GFAP. Vector 3 is a schematic diagram of the AAV-GFAP-CasRx-gRNA plasmid, which encodes CasRx and gRNA targeting negative regulatory genes (Sin3a, HDAC2, KDM1A, BAF53a, G9a, HuR, BrG1, EZH2, CDYL). Figure 8B is a schematic diagram of overexpressing various positive regulatory genes in the retina to transdifferentiate Muller glial cells into retinal ganglion cells or photoreceptor cells. Vector 1 is GFAP-driven mCherry, and vector 4 is GFAP-driven target gene expression, and the target gene is DPYSL2, BAF45b, SCF, CYP1B1, or BTRC.

[0229] Figure 9: Knockdown of negative regulatory genes in the mouse eye converts Müller glia into photoreceptors. Figure 9A shows the specific labeling of Müller glia by injection of a control AAV (AAV-GFAP-EGFP-P2A-Cre) into the retina of Ai9 mice. Yellow arrows (the arrowheads in Figure 9A are yellow arrows) indicate Müller glia specifically labeled with GFAP-EGFP-P2A-Cre. The cell bodies of Müller glia are located in the INL layer, and no red cells labeled with tdTomato are found in the ONL layer. Figures 9B-9J respectively show that different AAVs in the test group were injected subretinaally in mice (AAV-GFAP-mCherry+AAV-GFAP-CasRx-gRNA), where gRNAs targeted genes such as Sin3a, HDAC2, BAF53a, G9a, HuR, BrG1, EZH2, CDYL, and KDM1A, respectively. White arrows indicate tdTomato-positive cells in the ONL layer, and yellow arrows indicate tdTomato-positive Muller glial cells in the INL layer. The leftmost column of Figures 9A to 9J shows the results of mCherry labeling. The bright parts in the figures are red. The arrows in the INL column in Figures 9A to 9J are yellow arrows, and the arrows in the ONL column are white arrows.

[0230] Figure 10: Figures 10A and 10B show the conversion of Müller glial cells into photoreceptors following subretinal injection of the test AAV (AAV-GFAP-EGFP-P2A-Cre + AAV-GFAP-positive regulatory gene) in mice. The positive regulatory genes are DPYSL2 and BAF45b, respectively. White arrows indicate tdTomato-positive cells in the ONL layer, and yellow arrows indicate tdTomato-positive Müller glial cells in the INL layer. The scale bar is 50 μm. The leftmost column shows the results of mCherry labeling. The bright areas in the left column are red. Arrows in the INL column are yellow, and arrows in the ONL column are white.

[0231] Figure 11: Cytometry of Müller glial cells reprogrammed into photoreceptor cells.

[0232] Figure 12: Knockdown of Sin3a or KDM1A in the mouse eye converts Muller glial cells into rod cells. Figure 12A shows the result of injecting AAV-GFAP-EGFP-P2A-Cre+AAV-GFAP-CasRx into the retina of Ai9 mice. The white signal is rhodopsin staining, a rod-specific protein marker. The yellow arrow (the arrow in Figure 12A is a yellow arrow) indicates that tdTomato-labeled Muller glial cells are located in the INL layer, and there are no tdTomato-positive cells in the ONL layer. Figures 12B and 12C show the transdifferentiation results of the test group of AAVs targeting Sin3a and KDM1A (AAV-GFAP-mCherry+AAV-GFAP-CasRx-gRNA) injected subretina into Ai9 mice. The white arrows indicate that tdTomato-positive cells in the ONL layer are co-labeled with rhodopsin. The scale bar is 50 microns. The leftmost columns of Figures 12A to 12C are the results of mCherry labeling. The bright parts in the left columns are red, and the arrows in Figures 12B and C are white arrows.

[0233] Figure 13: Knockdown of Sin3a or EZH2 in the mouse eye converts Müller glia into cones. Figure 13A shows the results of injecting AAV-GFAP-EGFP-P2A-Cre into the retina of Ai9 mice. GFAP-EGFP-P2A-Cre specifically labels Müller glia, whose cell bodies are located in the INL layer. No red cells labeled with tdTomato were found in the ONL layer. The green signal is EGFP fluorescence expressed by GFAP-EGFP-P2A-Cre. Yellow arrows (the arrowheads in Figure 13A are yellow arrows) indicate Müller glia co-labeled with green and red signals. Figure 13B shows the results of injecting the control group AAV (AAV-GFAP-EGFP-P2A-Cre+AAV-GFAP-CasRx) into the retina of Ai9 mice. The white signal is the cone-specific protein marker m-CAR. The cyan arrows (the arrows in Figure 13B are cyan arrows) point to tdTomato-positive Muller glial cells. No tdTomato-positive cells were found in the ONL layer. Figures 13C and 13D are the results of injecting the test group AAV (AAV-GFAP-mCherry+AAV-GFAP-CasRx-gRNA) targeting Sin3a and EZH2 under the mouse retina, respectively. The white arrows (the arrows in Figures 13C and 13D are white arrows) indicate cells that are tdTomato-positive in the ONL layer and co-labeled with mCAR. The scale bar is 50 microns. The bright part in the mCherry column is red, the bright part in the EGFP column is green, and the bright part in the DAPI is blue.

[0234] Figure 14: Knockdown of negative regulatory genes in the mouse eye converts Muller glial cells into retinal ganglion cells. Figure 14A shows the result of injecting the control group AAV (AAV-GFAP-EGFP-P2A-Cre) into the retina of Ai9 mice. There is almost no labeled optic nerve in the optic nerve. Figures 14B to 14I respectively show the results of injecting the test group AAV (AAV-GFAP-mCherry + AAV-GFAP-CasRx-gRNA) under the retina of Ai9 mice to inhibit different negative regulatory genes, where gRNA targets HDAC2, BAF53a, G9a, HuR, BrG1, EZH2, CDYL, and KDM1A respectively.

[0235] Figure 15: Figures 15A to 15G show the conversion of Müller glia into retinal ganglion cells by overexpressing different positive regulatory genes in mouse eyes. The positive regulatory genes are DPYSL2, BAF45b, SCF, HuB, HuD, CYP1B1, and BTRC. The scale bar is 150 μm.

[0236] Figure 16: Statistical diagram of reprogramming of Müller glia into retinal ganglion cells.

[0237] Figure 17: Results of reprogramming Muller glial cells into photoreceptor cells in retinitis pigmentosa model mice. Figure 17A is a schematic diagram of the degeneration and death of rods and cones and the time points of AAV injection in retinitis pigmentosa model mice (Pde6b-KO). AAV injection was performed after all the retinal photoreceptor cells in Pde6b-KO mice died (P50), and retinal tissue was taken for analysis 50 days after injection; Figures 17B and 17C are retinal tissues taken from Pde6b-KO mice at 4 weeks and 6 weeks of age, respectively, and immunofluorescence staining was performed with the rod cell-specific marker Rhodopsin. No rod cells were observed at 4 weeks and 6 weeks. Figure 17D is the result of immunofluorescence staining of retinal tissue sections injected with the control group AAV (GFAP-CasRx+CBH-Pde6b). There are no Rhodopsin-positive cells in the ONL layer. Figures 17E to 17F show the immunofluorescence staining results of retinal tissue sections from the AAV-injected (GFAP-CasRx-gRNA + CBH-Pde6b) treatment groups. GRNA-targeted knockdown of EZH2 or KDM1A expression resulted in the reappearance of Rhodopsin-positive cells in the ONL layer. The bright color in the Rhodopsin column is green, and the bright color in the DAPI column is blue. Figure 17G shows the number of photoreceptor cells in the retinas of Pde6b-KO mice treated with different methods. The number of photoreceptor cells in the retinas of Pde6b-KO mice without AAV injection at 4 weeks and 6 weeks is shown. The number of photoreceptor cells in the EZH2 or KDM1A knockdown groups was significantly increased. Figure 17H ​​shows the thickness of the ONL layer in the retinas of Pde6b-KO mice treated with different methods. The thickness of the ONL layer in the retinas of Pde6b-KO mice without AAV injection at 4 weeks and 6 weeks is shown. The ONL layer was significantly thickened in the EZH2 or KDM1A knockdown groups. DETAILED DESCRIPTION

[0238] After extensive and in-depth research, the inventors unexpectedly discovered for the first time that inhibiting the expression or activity of one or more genes or their RNAs or proteins encoded by them in glial cells: RCOR1, RCOR2, RCOR3, Sin3a, Sin3b, HDAC1, HDAC2, KDM1A, PHF21A, BAF53a, G9a, USP14, HuR, BrG1, EZH2, CDYL, HMG20B, and / or enhancing the expression or activity of one or more genes or their RNAs or proteins encoded by them in glial cells: DPYSL2, BAF45b, SCF, HuB, HuC, HuD, CYP1B1, BTRC, can effectively induce the differentiation of glial cells into neurons, thereby treating neurological diseases associated with neuronal dysfunction or death. On this basis, the inventors completed the technical solution of the present application.

[0239] As used herein, the singular forms "a," "an," and "the" may include plural referents unless otherwise specified. For example, reference to "an inhibitor" may include a plurality of inhibitors, and reference to "the agent" includes reference to one or more agents and equivalents thereof known to those skilled in the art.

[0240] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and reagents similar or equivalent to those described herein can be used in the practice of the disclosed methods and compositions, exemplary methods and materials are now described.

[0241] Unless stated otherwise, herein, the use of "and" also includes "and / or." Similarly, "including" and "comprising" are interchangeable and are not intended to be limiting.

[0242] It should be further understood that while the term "comprising" is used in the description of various embodiments, those skilled in the art will understand that in some specific instances, the embodiments may alternatively be described using the language "consisting essentially of" or "consisting of."

[0243] In the present disclosure, degeneration of retinal ganglion cells (RGCs) is the main cause of permanent blindness. Transdifferentiation of Müller glial cells (MGs) into RGCs can help restore vision. The inventors found that by reducing (e.g., knocking down using the RNA-targeted CRISPR system CasRx) the gene or its RNA or its encoded protein in the retina of mature mice, MGs can be directly converted into RGCs. In addition, in a mouse model of retinal damage induced by NMDA (N-methyl-D-aspartate), RGCs converted from MGs achieved functional projections to the central visual area and improved visual function. Therefore, reducing negative regulatory genes (e.g., CasRx-mediated knockdown) would be a promising therapy for treating retinal diseases caused by neurodegeneration.

[0244] This application uses the recently characterized RNA-targeted CRISPR system CasRx to inhibit the gene or its RNA or its encoded protein.

[0245] As used herein, Müller glial cells (MG) are the main glial cells in the retina, and retinal ganglion cells (RGC) are nerve cells located in the innermost layer of the retina. Their dendrites are mainly connected to bipolar cells, and their axons extend to the optic nerve head to form the optic nerve.

[0246] Unless otherwise indicated, the terms "differentiation," "reprogramming," or "transdifferentiation" have the same meaning herein and may refer to the process of generating cells of a specific lineage (e.g., neuronal cells) from different types of non-neural cells (e.g., astrocytes) without intermediate differentiation.

[0247] As used herein, "contacting a negative modulator or a positive modulator with the non-neuronal cells" refers to placing a negative modulator or a positive modulator (e.g., a compound, a nucleic acid, a viral vector, etc.) in a position that allows it to contact the cells to produce "contacted" cells. Contacting can be accomplished using any suitable method. For example, in one embodiment, contacting is by adding a negative modulator or a positive modulator to a cell culture. Contacting can also be accomplished by injecting a negative modulator or a positive modulator, or a vector containing a negative modulator or a positive modulator, into a location in the body; or delivering a negative modulator or a positive modulator, or a vehicle containing a negative modulator or a positive modulator, to a location in the body so that the negative modulator or positive modulator "contacts" the targeted cell type.

[0248] In some embodiments, the effective amount of the negative modulator or the positive modulator is contacted with the non-neuronal cells in vivo to induce the non-neuronal cells to transdifferentiate into neurons in vitro. The positive modulator or the negative modulator is formulated into a medicament for in vivo administration to the nervous system, visual system, and auditory system, for example, in vivo administration to the striatum, substantia nigra, ventral tegmental area of ​​the midbrain, spinal cord, hypothalamus, dorsal midbrain, cerebral cortex, hippocampus, cerebellum, subretinal space, vitreous cavity, inner ear cochlea, and vestibule, preferably the striatum, subretinal space, subretinal space, and vitreous cavity.

[0249] In some embodiments, the effective amount of the negative regulator or the positive regulator is contacted with the non-neuronal cells in vitro to induce the non-neuronal cells to transdifferentiate into neurons in vitro. In some embodiments, the neurons obtained by in vitro contact can be formulated into a suitable cell therapy agent and administered to an individual in need thereof via a suitable administration method, such as intravenous infusion, in situ injection, etc.

[0250] The term "ONL" refers to the outer granule cell layer, the term "INL" refers to the inner granule cell layer, and the term "GCL" refers to the retinal ganglion cell layer.

[0251] As used herein, the term "non-neuronal cell" can refer to any type of cell that is not a neuron. In some embodiments, the non-neuronal cell is a stem cell, a progenitor cell, or a terminally differentiated cell.

[0252] In some embodiments, non-neuronal cells are cells of cell lineages (for example, hematopoietic lineages) other than neuronal lineages. In some embodiments, non-neuronal cells are cells of neuronal lineages but not neurons, for example, glial cells. In some embodiments, non-neuronal cells are terminally differentiated cells of non-neuron, such as, but not limited to, glial cells, fibroblasts, embryonic fibroblasts, epithelial cells, melanocytes, keratinocytes, adipocytes, hemocytes, bone marrow stromal cells, Langerhans cells, muscle cells, rectal cells or chondrocytes. In some embodiments, non-neuronal cells are from non-neuronal cell lines, such as, but not limited to, glioblastoma cell lines, Hela cell lines, NT2 cell lines, ARPE19 cell lines or N2A cell lines." cell lineage" or "pedigree" can represent the developmental history of tissue or organ from fertilized embryos. "Neuronal lineage" can refer to the developmental history from neural stem cells to mature neurons, including various stages along this process (called neurogenesis), such as but not limited to neural stem cells (neuroepithelial cells, radial glial cells), neural progenitor cells (e.g., interneuron precursors), neurons, astrocytes, oligodendrocytes, and microglia.

[0253] In some embodiments, the non-neuronal cell is a stem cell, such as an embryonic stem cell, a neural stem cell, or an induced pluripotent stem cell. In some embodiments, the non-neuronal cell is a progenitor cell, such as a neural progenitor cell or a neural precursor cell (such as a dopamine neural precursor cell).

[0254] In some embodiments, the non-neuronal cells are derived from the brain, preferably from the cerebrum, midbrain, cerebellum, brainstem, or spinal cord; more preferably, from the striatum or substantia nigra in the brain. Preferably, the non-neuronal cells are astrocytes derived from the brain, preferably from the striatum or substantia nigra. In some embodiments, the non-neuronal cells are derived from the eye, preferably from Müller glial cells from the eye.

[0255] The term "progenitor cell" refers to an intermediate cell present in adult tissues before differentiating into terminally differentiated cells. The differentiation of progenitor cells is usually clear.

[0256] The term "terminally differentiated cells," also known as terminal cells, refers to cells that have a specific function and a specific type and no longer differentiate, divide, or proliferate. Examples include glial cells, somatic cells, fibroblasts, red blood cells, mature epidermal cells, and muscle cells.

[0257] The term "stem cell" should be understood as an undifferentiated cell that has differentiation potential and proliferation capacity (particularly self-renewal capacity) but retains differentiation potential. Depending on the differentiation potential, stem cells include subpopulations such as pluripotent stem cells (PSCs), multipotent stem cells, unipotent stem cells, and embryonic stem cells. In some embodiments, the stem cell can be an embryonic stem cell, a neural stem cell, or an induced pluripotent stem cell.

[0258] As used herein, the term "pluripotent stem cell" (PSC) refers to a stem cell that can be cultured in vitro and has the ability to differentiate into any cell lineage belonging to the three germ layers (ectoderm, mesoderm, endoderm). PSCs can be induced from fertilized eggs, cloned embryos, germ stem cells, stem cells in tissues, somatic cells, etc. Examples of PSCs include embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs or iPS), embryonic germ cells (EG cells), etc.

[0259] As used herein, the term "induced pluripotent stem cells" (iPS or iPSC) can be generated directly from adult cells by reprogramming. Adult cells can be converted into PSCs by introducing the products of a specific set of pluripotency-associated genes.

[0260] As used herein, the term "neural precursor cells" refers to cells that have the potential to develop into neurons and are in a neuron precursor state.

[0261] Term " neuron " can have the general meaning that those skilled in the art will understand.In some embodiments, neuron can refer to can receive, process and transmit information by electric signal (for example, membrane potential discharge) and chemical signal (for example, the synaptic transmission of neurotransmitter) electrically excitable cell.As understood by those skilled in the art, the chemical signal (for example, based on the release and identification of neurotransmitter) transduced between neurons can occur by the special connection called synapse.In some embodiments, described neuron is dopaminergic neuron, retinal ganglion cell, photoreceptor cell, 5-HT neuron, NE neuron, ChAT neuron, motor neuron, GABA neuron, glutamatergic neuron, spinal neuron, spinal motor neuron, spinal sensory neuron, bipolar cell, amacrine cell, cochlear neuron, pyramidal neuron, interneuron, medium spiny neuron, Purkinje cell, granule cell, olfactory sensory neuron or periglomerular cell or its combination.

[0262] As used herein, the terms "protein," "peptide," and "polypeptide" may refer to an amino acid polymer or a group of two or more interacting or bound amino acid polymers and have the same meaning.

[0263] As used herein, the terms "nucleic acid" and "polynucleotide" have the same meaning and may refer to a nucleic acid molecule containing one or more nucleotides.

[0264] As used herein, the terms "dopaminergic neuron" and "dopamine neuron" have the same meaning.

[0265] As used herein, the term "target gene" refers to a gene regulated by a "negative regulator" or a "positive regulator," for example: RCOR1, RCOR2, RCOR3, Sin3a, Sin3b, HDAC1, HDAC2, KDM1A, PHF21A, BAF53a, G9a, USP14, HuR, BrG1, EZH2, CDYL, HMG20B, DPYSL2, BAF45b, SCF, HuB, HuC, HuD, CYP1B1, or BTRC.

[0266] The term "photoreceptor" refers to a type of neuroepithelial cell in the retina that transmits light for vision, including rods and cones. Some diseases such as retinitis pigmentosa, macular degeneration, and diabetic complications can cause the death of photoreceptor cells.

[0267] The term "ganglion cell" refers to neurons located in the terminal segment of the retina. Many eye diseases, such as ischemic retinopathy and glaucoma, can lead to the death of retinal ganglion cells, which can also cause permanent blindness.

[0268] As used herein, Müller glial cells (MG cells) are the main glial cells in the retina, and retinal ganglion cells (RGCs) are nerve cells located in the innermost layer of the retina. Their dendrites are mainly connected to bipolar cells, and their axons extend to the optic nerve head to form the optic nerve.

[0269] The term "carrier" refers to a tool capable of transporting exogenous nucleic acids into cells, which can be nucleic acids, proteins, etc. When the carrier is present in an appropriate environment, the carrier can direct the expression of one or more proteins encoded by one or more genes carried by the carrier or the microRNA encoded by the polynucleotides carried by the carrier. In some embodiments, the carrier can be used to introduce the polynucleotides provided herein. In this article, the carrier can be a variety of different forms of carriers. In some embodiments, the carrier can be a viral vector, a plasmid vector, a mini-circle vector, a linear DNA vector, a dog bone vector (doggybone vectors), a liposome, a nanoparticle, an exosome, an extracellular vesicle, a cationic polymer (such as PEI) or a virus-like particle, etc.

[0270] The term "viral vector" refers to a virally derived nucleic acid that can transport another nucleic acid into a cell. When a viral vector is present in an appropriate environment, it can direct the expression of one or more proteins encoded by one or more genes carried by the vector or microRNAs encoded by polynucleotides carried by the vector. Examples of viral vectors include, but are not limited to, retroviral vectors, adenoviral vectors, lentiviral vectors, poxvirus vectors, herpesvirus vectors, and adeno-associated virus vectors.

[0271] In some embodiments, the nanoparticle carrier may include polymer-based nanoparticles, aminoester-based nanoparticles, or metal nanoparticles.

[0272] The carrier that this paper provided can be used to send the polynucleotide compositions that this paper provided.In some cases, use a single carrier to deliver at least about 2, 3, 4 or 5 polynucleotides at the most.In some cases, use a single carrier to deliver at least about 2, 3, 4 or 5 different polynucleotides at the most.In some cases, use a single carrier to deliver at least about 2, 3, 4 or 5 identical polynucleotides at the most.

[0273] In some embodiments, the vector can deliver DNA (such as double-stranded DNA or single-stranded DNA), and can also deliver RNA. In some cases, the RNA can include base modifications. The vector can include a recombinant vector. The vector can be a vector modified from a naturally occurring vector. The vector can include at least a portion of a non-naturally occurring vector. Any vector can be used.

[0274] In this article, "astrocyte" may refer to the characteristic star-shaped glial cells in the brain and spinal cord. It is clear to those skilled in the art that the characteristics of astrocytes may be star-shaped, expressing markers such as glial fibrillary acidic protein (GFAP) and aldehyde dehydrogenase 1 family member L1 (ALDH1L1), excitatory amino acid transporter 1 / glutamate aspartate transporter (EAAT1 / GLAST), glutamine synthetase, S100β or excitatory amino acid transporter 1 / glutamate transporter 1 (EAAT2 / GLT-1), together with endothelial cells, participate in the blood-brain barrier, transmitter uptake and release, regulate the ion concentration in the extracellular space, respond to neuronal damage and participate in nervous system repair and metabolic support surrounding neurons. In certain embodiments of the present invention, astrocytes may refer to non-neuronal cells in the nervous system that express glial fibrillary acidic protein (GFAP), aldehyde dehydrogenase 1 family member L1 (ALDH1L1) or both. In certain embodiments, astrocytes may refer to non-neuronal cells in the nervous system that express a transgene (eg, red fluorescent protein (RFP), Cre recombinase) driven by the glial fibrillary acidic protein (GFAP) promoter.

[0275] In some embodiments, the glial cells useful in the methods provided herein are glial cells isolated from the brain. In some embodiments, the astrocytes useful in the methods provided herein are star-shaped glial cells in the brain or spinal cord.

[0276] The "effective amount" of the negative regulator or the positive regulator in contact with the non-neuronal cells refers to the amount of the negative regulator or the positive regulator that can convert the non-neuronal cells into neurons, or the amount of the carrier containing the negative regulator or the positive regulator.

[0277] As used herein, "active fragment," "functional fragment," and "functional fragment" have the same meaning and refer to a truncated fragment of a gene or protein that has the same or similar function as the full-length gene or protein. For example, a "functional fragment of SEQ ID NO: 36" refers to a truncated fragment of SEQ ID NO: 36 that has a similar function as the sequence set forth in SEQ ID NO: 36. This truncated fragment can exert the same or similar positive regulatory effect as SEQ ID NO: 36, promoting the transdifferentiation of non-neuronal cells into neurons.

[0278] As used herein, the inhibitor of a negative regulatory gene can be an RNA interfering agent that contains RNA and mediates the targeted cleavage of RNA transcripts through the RNA-induced silencing complex (RISC) pathway. RNA interfering agents guide the sequence-specific degradation of mRNA through the process of RNA interference (RNAi). RNA interfering agents can inhibit the expression of one or more negative regulatory genes in cells. RNA interfering agents include but are not limited to "small interfering RNA (siRNA)", "siRNA prepared by endoribonuclease (e-siRNA)", "short hairpin RNA (shRNA)" and "small time-regulated RNA" (stRNA)", "cut siRNA (d-siRNA)" and aptamers, oligonucleotides and other synthetic nucleic acids containing at least one uracil base. In some embodiments, such RNA interfering agents are delivered by a carrier, such as, but not limited to, a replication-deficient or replication-competent viral vector (e.g., adenovirus, lentivirus, gamma retrovirus, adeno-associated virus, etc.).

[0279] In some embodiments, the present invention provides methods for generating neurons in vivo. Exemplary methods include administering a positive or negative modulator to a region of a subject's nervous system, such as the brain, eye, or spinal cord, and allowing non-neuronal cells to reprogram into functional neurons.

[0280] The term "expression vector" refers to a vector that adds expression elements (such as a promoter, RBS, GOI, and terminator) to the basic cloning vector framework to enable the expression of the target gene. Construction of an expression vector enables the target gene to be expressed and function in recipient cells.

[0281] "Negative regulatory genes" and "negative regulators"

[0282] As used herein, "negative regulatory gene" refers to a gene that can increase, promote or improve the transdifferentiation of non-neuronal cells into neurons when the expression of the gene, the expression of its mRNA, or the expression of the protein encoded by the gene is reduced or inhibited, for example: RCOR1, RCOR2, RCOR3, Sin3a, Sin3b, HDAC1, HDAC2, KDM1A, PHF21A, BAF53a, G9a, USP14, HuR, BrG1, EZH2, CDYL, HMG20B, etc.

[0283] As used herein, a "negative regulator" refers to a regulator that can reduce or inhibit the gene expression, mRNA expression, or expression of the protein encoded by a "negative regulatory gene." In some embodiments, a negative regulator can be a gene editing tool or epigenetic regulation tool that can reduce the expression of a negative regulatory gene; it can also be an inhibitor of a negative regulatory gene, an inhibitor of negative regulatory gene activity, or an activator that degrades the protein encoded by the negative regulatory gene.

[0284] In some embodiments, a "negative regulator" can be an inhibitory antibody of a negatively regulated gene; or a small molecule inhibitor of a negatively regulated gene; or an inhibitory mRNA, microRNA, siRNA, shRNA, antisense oligonucleotide, binding protein or protein domain, polypeptide, nucleic acid aptamer, or PROTAC of a negatively regulated gene; or an inhibitory binding protein or ligand of a negatively regulated gene.

[0285] In some embodiments, the gene editing tool used as a "negative regulator" can be a CRISPR gene editing tool, which includes a gene editing protein and a guide RNA (gRNA) that targets the negatively regulated gene. In specific embodiments, the gene editing protein can be a DNA editing protein, such as Cas9, or an RNA editing protein, such as Cas13.

[0286] In some specific embodiments, the gene editing tool serving as a "negative regulator" may be a gene editing tool comprising zinc finger nucleases, or a TALENs (transcription activator-like (TAL) effector nucleases)) gene editing tool.

[0287] In some embodiments, "negative regulators" may be epigenetic regulatory tools. Epigenetic regulatory tools refer to proteins, genes, small molecules, and the like that regulate the content and function of intracellular nucleic acids or proteins through epigenetic modifications (such as methylation, acetylation, phosphorylation, and changes in chromatin conformation) without altering the nucleotide sequence of the target gene. For example, epigenetic regulatory proteins such as KRAB and DNMT3A may be fused to a mutant CRISPR / Cas system.

[0288] "Positive regulatory genes" and "positive regulators"

[0289] As used herein, "positive regulatory gene" refers to a gene that can enhance, promote or improve the transdifferentiation of non-neuronal cells into neurons when the expression of the gene, the expression of its mRNA, or the expression of the protein encoded by the gene is increased or activated, for example: DPYSL2, BAF45b, SCF, HuB, HuC, HuD, CYP1B1, or BTRC.

[0290] As used herein, a "positive regulator" refers to a regulator that can increase or activate the gene expression, mRNA expression, or gene-encoded protein expression of a "positive regulatory gene." In some embodiments, the positive regulator can be an epigenetic regulatory tool that can increase the expression of a positive regulatory gene, an activator of positive regulatory gene expression, an inhibitor of degradation of a protein encoded by a positive regulatory gene, a stabilizer of positive regulatory gene mRNA, or an exogenous positive regulatory gene or a functional fragment of a positive regulatory gene.

[0291] In some embodiments, a "positive regulator" can be: an agonistic antibody of a positively regulated gene; or a small molecule agonist of a positively regulated gene; or an agonistic binding protein or ligand of a positively regulated gene; or an inhibitor of a competitive gene of a positively regulated gene.

[0292] Diseases associated with loss of neuronal function or neuronal death

[0293] In the present disclosure, the disease associated with neuronal dysfunction or neuronal death can be Parkinson's disease, a visual system disease associated with RGC or photoreceptor cell dysfunction or death, Alzheimer's disease, brain injury, Huntington's disease, epilepsy, depression, sleep disorders, cerebral ischemia, motor neuron disease, amyotrophic lateral sclerosis, spinal muscular atrophy, ataxia, PolyQ disease, schizophrenia, addiction, Pick's disease, blindness caused by RGC or photoreceptor cell damage or death, macular degeneration, retinitis pigmentosa, deafness, night blindness, color blindness, hereditary blindness, congenital amaurosis, etc. In some embodiments, the disease associated with neuronal dysfunction or neuronal death is preferably a visual system disease associated with Parkinson's disease and RGC or photoreceptor cell dysfunction or death, such as: a disease associated with dopamine neuron dysfunction or death, and a visual disorder associated with optic ganglion or photoreceptor cell loss or death.

[0294] The visual system disease associated with loss of function or death of RGC or photoreceptor cells is preferably selected from: vision impairment caused by death of RGC cells or photoreceptor cells, glaucoma, age-related RGC lesions, optic nerve damage, age-related macular degeneration (AMD), diabetic retinopathy, retinal ischemia or hemorrhage, Leber hereditary optic neuropathy, or a combination thereof; the visual system disease associated with loss of function or death of photoreceptor cells is preferably selected from: degeneration or death of photoreceptor cells caused by damage or degenerative lesions, macular degeneration, retinitis pigmentosa, diabetes-related blindness, night blindness, color blindness, hereditary blindness, congenital amaurosis, or a combination thereof.

[0295] astrocytes

[0296] Astrocytes are the most numerous cell type in the mammalian brain. They perform numerous functions, including biochemical support (e.g., forming the blood-brain barrier), providing nutrients to neurons, maintaining extracellular ion balance, and participating in repair and scar formation after brain and spinal cord injury. Astrocytes can be divided into two types based on the content of glial filaments and the shape of their processes: fibrous astrocytes, which are primarily distributed in the white matter of the brain and spinal cord, have slender processes with few branches and contain abundant glial filaments in their cytoplasm; and protoplasmic astrocytes, which are primarily distributed in the gray matter and have short, stubby processes with numerous branches.

[0297] Astrocytes that can be used in the present disclosure are not particularly limited, and include various astrocytes derived from the mammalian central nervous system, such as from the striatum, substantia nigra, ventral tegmental area, hypothalamus, spinal cord, dorsal midbrain or cerebral cortex, preferably from the striatum and substantia nigra.

[0298] Neurons

[0299] In the present disclosure, a functional neuron may refer to a neuron that is capable of sending or receiving information via chemical or electrical signals. In some embodiments, a functional neuron exhibits one or more functional characteristics of mature neurons present in a normal nervous system, including but not limited to: excitability (e.g., the ability to exhibit an action potential, such as a rapid rise and subsequent fall) (voltage across the cell membrane or membrane potential), formation of synaptic connections with other neurons, presynaptic neurotransmitter release, and postsynaptic responses (e.g., excitatory postsynaptic current or inhibitory postsynaptic current).

[0300] In some embodiments, a functional neuron is characterized in that it expresses one or more markers of functional neurons, including but not limited to synaptophysin, synaptophysin, glutamate decarboxylase 67 (GAD67), glutamate decarboxylase 65 (GAD65), parvalbumin, dopamine- and cAMP-regulated neuronal phosphoprotein 32 (DARPP32), vesicular glutamate transporter 1 (vGLUT1), vesicular glutamate transporter 2 (vGLUT2), acetylcholine, tyrosine hydroxylase (TH), dopamine, vesicular GABA transporter (VGAT), and gamma-aminobutyric acid (GABA).

[0301] In some embodiments, the functional neurons can be dopaminergic neurons, 5-HT neurons, NE neurons, ChAT neurons, GABA neurons, glutamatergic neurons, motor neurons, photoreceptor cells (such as rods and cones), retinal ganglion cells (RGCs), cochlear nerve cells (such as cochlear spiral ganglion cells and vestibular neurons), or medium spiny neurons (MSNs) or a combination thereof, preferably dopaminergic neurons, retinal ganglion cells and photoreceptor cells.

[0302] In some embodiments, the functional neurons are mammalian neurons, such as neurons of humans, non-human primates, rats, or mice.

[0303] Dopaminergic neurons or dopamine neurons

[0304] Dopaminergic neurons are neurons that contain and release dopamine (DA) as a neurotransmitter. Dopamine belongs to the catecholamine neurotransmitter family and plays an important biological role in the central nervous system. Dopaminergic neurons in the brain are primarily concentrated in the substantia nigra pars compacta (SNc), ventral tegmental area (VTA), hypothalamus, and periventricular cortex of the midbrain. Numerous studies have confirmed that dopaminergic neurons are closely associated with various human diseases, the most prominent of which is Parkinson's disease.

[0305] gene editing tools

[0306] Gene editing tools refer to the process of modifying the target gene through gene editing technology, including the insertion, deletion or replacement of the gene, thereby changing its genetic information and phenotypic characteristics.

[0307] In the present disclosure, available gene editing tools include, but are not limited to, CRISPR gene editing tools (CRISPR / Cas system), zinc finger nuclease gene editing tools (ZFN system), and TALENs (transcription activator-like (TAL) effector nucleases)) gene editing tools (TALEN system).

[0308] In some embodiments, the gene editing tool comprises a DNA gene editing tool or an RNA gene editing tool.

[0309] In a preferred embodiment, the gene editing tool of the present disclosure is a CRIPSR gene editing tool. In some embodiments, the CRIPSR gene editing tool includes a nucleic acid encoding a Cas protein (also referred to herein as a Cas enzyme) or a functional domain of a Cas protein, and a gRNA targeting a target gene. The gRNA can guide the Cas protein to target the target gene and perform gene editing.

[0310] In some embodiments, the Cas protein is a protein or mutant thereof of a family such as Cas9, Cas12 (e.g., Cas12a, Cas12b, Cas12c, Cas12d, etc.), Cas13, Cas 14, CasX, or CasY. In some embodiments, the Cas protein is Cas13a, Cas13d, Cas13X, Cas13a, Cas13b (e.g., Cas13b-t1, Cas13b-t2, Cas13b-t3, etc.), Cas13c, or Cas13Y. In some embodiments, the Cas13d is CasRx.

[0311] In a preferred embodiment, the gene editing tool can reduce or silence the expression of the negative regulatory genes involved in this application.

[0312] In some cases, gene editing tools can be delivered via a vector, for example, by delivering the gRNA and gene editing protein (eg, CasRx) provided herein via an adeno-associated virus.

[0313] Cas proteins

[0314] In the present disclosure, the nucleotides of the Cas protein can be obtained by genetic engineering techniques, such as genome sequencing, polymerase chain reaction (PCR), etc., and its amino acid sequence can be derived from the nucleotide sequence. The sources of the wild-type Cas protein include but are not limited to: Ruminococcus laefaciens, Streptococcus pyogenes, Staphylococcus aureus, Acidaminococcus sp, and Lachnospiraceaeacterium.

[0315] In a preferred embodiment of the present disclosure, the Cas protein is capable of editing DNA.

[0316] In a preferred embodiment of the present disclosure, the Cas protein is capable of editing RNA. For example, the Cas protein can be an RNA-targeted gene editing protein such as Cas13d, Cas13e, Cas13a, Cas13b, Cas13c, and Cas13f.

[0317] Peptides, proteins, proteins, and polynucleotides

[0318] In the present disclosure, the terms "protein of the present disclosure," "protein," "polypeptide," and "protein" are used interchangeably and may refer to proteins or polypeptides having the amino acid sequence of RCOR1, RCOR2, RCOR3, Sin3a, Sin3b, HDAC1, HDAC2, KDM1A, PHF21A, BAF53a, G9a, USP14, HuR, BrG1, EZH2, CDYL, HMG20B, BTRC, CYP1B1, DPYSL2, BAF45b, SCF, HuB, HuC, or HuD. These include proteins with or without an initiator methionine. In addition, the term also includes the full-length proteins and fragments thereof. The proteins referred to in the present disclosure include their complete amino acid sequences, secreted proteins, mutants thereof, and functionally active fragments thereof.

[0319] RCOR1, RCOR2, and RCOR3 stand for REST corepressor 1, REST corepressor 2, and REST corepressor 3, and they share similar functions. HDAC1 stands for histone deacetylase 1, and HDAC2 stands for histone deacetylase 2, and they share similar functions. KDM1A stands for lysine-specific demethylase 1A, also known as lysine-specific histone demethylase 1A (LSD1). Sin3a (SIN3 transcriptional regulatory protein family member A) and Sin3b (SIN3 transcriptional regulatory protein family member B) share similar functions.

[0320] In the present disclosure, the terms "gene", "polynucleotide" and "nucleic acid" are used interchangeably and may refer to a nucleic acid sequence having the nucleotide sequence of RCOR1, RCOR2, RCOR3, Sin3a, Sin3b, HDAC1, HDAC2, KDM1A, PHF21A, BAF53a, G9a, USP14, HuR, BrG1, EZH2, CDYL, HMG20B, CYP1B1, BTRC, DPYSL2, BAF45b, SCF, HuB, HuC or HuD. The genomic length of the human RCOR1 gene is 173980bp (NCBI GenBank accession number is 23186). The genomic length of the mouse RCOR1 gene is 76536bp (NCBI GenBank accession number is 217864). The genomic length of the human RCOR2 gene is 5935bp (NCBI GenBank accession number is 283248). The full-length genome of the mouse RCOR2 gene is 7832 bp (NCBI GenBank accession number is 104383). The full-length genome of the human RCOR3 gene is 57020 bp (NCBI GenBank accession number is 55758). The full-length genome of the mouse RCOR3 gene is 39526 bp (NCBI GenBank accession number is 214742). The full-length genome of the human Sin3a gene is 86441 bp (NCBI GenBank accession number is 25942). The full-length genome of the mouse Sin3a gene is 56382 bp (NCBI GenBank accession number is 20466). The full-length genome of the human Sin3b gene is 50958 bp (NCBI GenBank accession number is 23309). The full-length genome of the mouse Sin3b gene is 34934 bp (NCBI GenBank accession number is 20467). The full-length genome of the human HDAC1 gene is 41546 bp (NCBI GenBank accession number is 3065). The mouse HDAC1 gene has a genomic length of 26,543 bp (NCBI GenBank accession number 433759). The human HDAC2 gene has a genomic length of 38,121 bp (NCBI GenBank accession number 3066). The mouse HDAC2 gene has a genomic length of 27,593 bp (NCBI GenBank accession number 15182). The human KDM1A gene has a genomic length of 64,249 bp (NCBI GenBank accession number 23028). The mouse KDM1A gene has a genomic length of 52,284 bp (NCBI GenBank accession number 99982). The human PHF21A gene has a genomic length of 192,136 bp (NCBI GenBank accession number 51317).The full-length genome of the mouse PHF21A gene is 180,916 bp (NCBI GenBank accession number is 192285). The full-length genome of the human BAF53a gene is 25,482 bp (NCBI GenBank accession number is 86). The full-length genome of the mouse BAF53a gene is 18,428 bp (NCBI GenBank accession number is 56456). The full-length genome of the human G9a gene is 17,940 bp (NCBI GenBank accession number is 10919). The full-length genome of the mouse G9a gene is 15,624 bp (NCBI GenBank accession number is 110147). The full-length genome of the human USP14 gene is 56,073 bp (NCBI GenBank accession number is 9097). The full-length genome of the mouse USP14 gene is 36,535 bp (NCBI GenBank accession number is 59025). The full-length genome of the human HuR gene is 47,069 bp (NCBI GenBank accession number is 1994). The full-length genome of the mouse HuR gene is 40,324 bp (NCBI GenBank accession number is 15568). The full-length genome of the human BrG1 gene is 101,279 bp (NCBI GenBank accession number is 6597). The full-length genome of the mouse BrG1 gene is 88,150 bp (NCBI GenBank accession number is 20586). The full-length genome of the human EZH2 gene is 76,971 bp (NCBI GenBank accession number is 2146). The full-length genome of the mouse EZH2 gene is 65,102 bp (NCBI GenBank accession number is 14056). The full-length genome of the mouse CDYL gene is 214,247 bp (NCBI GenBank accession number is 12593). The full-length genome of the human CDYL gene is 249,407 bp (NCBI GenBank accession number is 9425). The full-length genome of the human HMG20B gene is 6,168 bp (NCBI GenBank accession number is 10362). The mouse HMG20B gene has a genomic length of 4898 bp (NCBI GenBank Accession No. 15353). The human DPYSL2 gene has a genomic length of 144,145 bp (NCBI GenBank Accession No. 1808). The mouse DPYSL2 gene has a genomic length of 108,178 bp (NCBI GenBank Accession No. 12934). The human BAF45b gene has a genomic length of 18,690 bp (NCBI GenBank Accession No. 8193). The mouse BAF45b gene has a genomic length of 13,652 bp (NCBI GenBank Accession No. 29861). The human SCF gene has a genomic length of 87,679 bp (NCBI GenBank Accession No. 4254).The full-length genome of the mouse SCF gene is 84,802 bp (NCBI GenBank accession number is 17311). The full-length genome of the human HuB gene is 160,503 bp (NCBI GenBank accession number is 1993). The full-length genome of the mouse HuB gene is 188,638 bp (NCBI GenBank accession number is 15569). The full-length genome of the human HuC gene is 29,721 bp (NCBI GenBank accession number is 1995). The full-length genome of the mouse HuC gene is 37,142 bp (NCBI GenBank accession number is 15571). The full-length genome of the human HuD gene is 155,718 bp (NCBI GenBank accession number is 1996). The full-length genome of the mouse HuD gene is 148,212 bp (NCBI GenBank accession number is 15572). The full-length genome of the human CYP1B1 gene is 8,643 bp (NCBI GenBank accession number is 1545). The mouse CYP1B1 gene has a full genomic length of 8122 bp (NCBI GenBank accession number 13078). The human BTRC gene has a full genomic length of 203,506 bp (NCBI GenBank accession number 8945). The mouse BTRC gene has a full genomic length of 169,664 bp (NCBI GenBank accession number 12234).

[0321] Human and mouse RCOR1 share 88.51% similarity at the DNA level and 92.39% similarity in protein sequence. Human and mouse RCOR2 share 89.26% similarity at the DNA level and 97.51% similarity in protein sequence. Human and mouse RCOR3 share 92.38% similarity at the DNA level and 96.39% similarity in protein sequence. Human and mouse Sin3a share 90.91% similarity at the DNA level and 98.04% similarity in protein sequence. Human and mouse Sin3b share 86.61% similarity at the DNA level and 87.91% similarity in protein sequence. Human and mouse HDAC1 share 90.85% similarity at the DNA level and 99.38% similarity in protein sequence. Human and mouse HDAC2 share 91.62% similarity at the DNA level and 99.59% similarity in protein sequence. Human and mouse KDM1A share 89.75% similarity at the DNA level and 97.72% similarity in protein sequence. Human and mouse PHF21A share 92.88% similarity at the DNA level and 94.20% similarity in protein sequence. Human and mouse BAF53a share 88.60% similarity at the DNA level and 98.84% similarity in protein sequence. Human and mouse G9a share 87.74% similarity at the DNA level and 94.75% similarity in protein sequence. Human and mouse USP14 share 91.31% similarity at the DNA level and 96.76% similarity in protein sequence. Human and mouse HuR share 91.03% similarity at the DNA level and 98.47% similarity in protein sequence. Human and mouse BrG1 share 89.00% similarity at the DNA level and 95.47% similarity in protein sequence. Human and mouse EZH2 share 91.98% similarity at the DNA level and 97.60% similarity in protein sequence. Human and mouse CDYL share 86.77% similarity at the DNA level and 93.12% similarity in protein sequence. Human and mouse HMG20B share 85.53% similarity at the DNA level and 93.69% similarity in protein sequence. Human and mouse DPYSL2 share 91.05% similarity at the DNA level and 97.64% similarity in protein sequence. Human and mouse BAF45b share 93.39% similarity at the DNA level and 97.69% similarity in protein sequence. Human and mouse SCF share 89.44% similarity at the DNA level and 82.78% similarity in protein sequence. Human and mouse HuB share 95.46% similarity at the DNA level and 99.72% similarity in protein sequence. Human and mouse HuC share 91.94% similarity at the DNA level and 99.46% similarity in protein sequence. Human and mouse HuD share 94.64% similarity at the DNA level and 99.21% similarity in protein sequence. Human and mouse CYP1B1 share 82.96% similarity at the DNA level and 81.03% similarity in protein sequence.Human and mouse BTRC share 90.87% similarity at the DNA level and 98.68% similarity at the protein level. It is understood that nucleotide substitutions within codons are acceptable when encoding the same amino acid. Furthermore, it is understood that nucleotide substitutions are also acceptable when resulting in conservative amino acid substitutions.

[0322] When an amino acid fragment is obtained, the nucleic acid sequence encoding it can be constructed according to the fragment, and specific probes can be designed according to the nucleotide sequence. The full-length nucleotide sequence or its fragment can usually be obtained by PCR amplification, recombination or artificial synthesis. For PCR amplification, primers can be designed according to the nucleotide sequence disclosed in this disclosure, especially the open reading frame sequence, and the relevant sequence can be amplified using a commercially available cDNA library or a cDNA library prepared by conventional methods known to those skilled in the art as a template. When the sequence is long, it is often necessary to carry out two or more PCR amplifications, and then the fragments amplified each time are spliced ​​together in the correct order.

[0323] Once the relevant sequence is obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, then transferring it into cells, and then isolating the relevant sequence from the propagated host cells by conventional methods.

[0324] In addition, the sequences can also be synthesized by artificial synthesis, especially when the fragment length is shorter. Usually, a long fragment can be obtained by synthesizing multiple small fragments and then connecting them.

[0325] Currently, DNA sequences encoding proteins of the present disclosure (or fragments or derivatives thereof) can be obtained entirely through chemical synthesis, which can then be introduced into various existing DNA molecules (such as vectors) and cells known in the art.

[0326] The polynucleotide sequences of the present disclosure can be used to express or produce recombinant polypeptides using conventional recombinant DNA techniques. Generally, the following steps are involved:

[0327] (1) Transforming or transducing a suitable host cell with a polynucleotide (or variant) encoding a polypeptide of the present disclosure, or with a recombinant expression vector containing the polynucleotide;

[0328] (2) Host cells cultured in a suitable culture medium;

[0329] (3) Isolate and purify proteins from culture medium or cells.

[0330] In the present disclosure, polynucleotide sequences can be inserted into recombinant expression vectors. In short, any plasmid or vector can be used as long as it can replicate and be stable in the host. An important feature of an expression vector is that it usually contains a replication origin, a promoter, a marker gene, and a translation control element.

[0331] Methods well known to those skilled in the art can be used to construct expression vectors containing the coding DNA sequence of RCOR1, RCOR2, RCOR3, Sin3a, Sin3b, HDAC1, HDAC2, KDM1A, PHF21A, BAF53a, G9a, USP14, HuR, BrG1, EZH2, CDYL, CYP1B1, BTRC, HMG20B, DPYSL2, BAF45b, SCF, HuB, HuC or HuD and suitable transcription / translation control signals. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc. The dna sequence dna can be effectively connected to a suitable promoter in the expression vector to guide mRNA synthesis. The expression vector also includes a ribosome bind site and a transcription terminator for translation initiation.

[0332] In addition, the expression vector preferably contains one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green fluorescent protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for Escherichia coli.

[0333] A vector containing the above-mentioned appropriate DNA sequence and an appropriate promoter or control sequence can be used to transform an appropriate host cell to enable it to express the protein.

[0334] Host cells can be prokaryotic cells, such as bacterial cells; lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples include: Escherichia coli, Streptomyces bacteria cells; fungal cells, such as yeast; plant cells; insect cells; animal cells, etc.

[0335] Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism such as Escherichia coli, competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated with CaCl2, using procedures well known in the art. Another method is to use MgCl2. If desired, transformation can also be performed using electroporation. When the host is a eukaryotic organism, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0336] The obtained transformants can be cultured with conventional methods to express the polypeptide encoded by the gene of the present disclosure. Depending on the host cell used, the culture medium used can be selected from various conventional culture media. Culture is carried out under conditions suitable for the growth of the host cells. After the host cells grow to an appropriate cell density, the selected promoter is induced using a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.

[0337] The recombinant polypeptide in the above method can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If necessary, the recombinant protein can be isolated and purified by various separation methods utilizing its physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to, conventional renaturation treatment, treatment with a protein precipitant (salting out method), centrifugation, osmotic sterilization, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC), and various other liquid chromatography techniques and combinations of these methods.

[0338] Adeno-associated virus

[0339] Because adeno-associated virus (AAV) is smaller than other viral vectors, has no pathogenicity, and can transfect both dividing and non-dividing cells, gene therapy methods based on AAV vectors for genetic diseases have received widespread attention.

[0340] Adeno-associated virus (AAV), also known as adeno-associated virus, belongs to the genus Dependivirus in the family Parvoviridae. It is the simplest single-stranded DNA defective virus discovered to date and requires a helper virus (usually an adenovirus) for replication. It encodes the cap and rep genes, located within the inverted repeats (ITRs) at its two terminals. The ITRs play a crucial role in viral replication and packaging. The cap gene encodes the viral capsid protein, while the rep gene is involved in viral replication and integration. AAV can infect a variety of cell types.

[0341] Recombinant adeno-associated virus (rAAV) vectors, derived from the non-pathogenic wild-type adeno-associated virus, are considered one of the most promising gene transfer vectors due to their safety, broad host cell range (dividing and non-dividing cells), low immunogenicity, and prolonged in vivo expression of foreign genes. They are widely used in gene therapy and vaccine research worldwide. After more than a decade of research, the biological properties of rAAV have been thoroughly understood, particularly with extensive data on their efficacy in various cell types, tissues, and in vivo experiments. In medical research, rAAV has been used in gene therapy studies for a variety of diseases (both in vitro and in vivo). As a unique gene transfer vector, it is also widely used in gene function studies, disease model development, and the generation of knockout mice.

[0342] In a preferred embodiment of the present disclosure, the vector is a recombinant AAV vector. AAV is a relatively small DNA virus that can integrate into the genome of the cells they infect in a stable and site-specific manner. They are able to infect a large range of cells without any effect on cell growth, morphology or differentiation, and they do not seem to be involved in human pathology. The AAV genome has been cloned, sequenced and characterized. AAV contains an inverted terminal repeat (ITR) region of approximately 145 bases at each end, which serves as the origin of viral replication. The rest of the genome is divided into two important regions with encapsidation functions: the left part of the genome containing the rep genes involved in viral replication and viral gene expression; and the right part of the genome containing the cap gene encoding the viral capsid protein.

[0343] AAV vectors can be prepared using standard methods in the art. Adeno-associated viruses of any serotype are suitable. Methods for purifying vectors can be found in, for example, U.S. Patent Nos. 6,566,118, 6,989,264, and 6,995,006, the disclosures of which are incorporated herein by reference in their entirety. The preparation of hybrid vectors is described, for example, in PCT Application No. PCT / US2005 / 027091, the disclosures of which are incorporated herein by reference in their entirety. The use of vectors derived from AAV for transporting genes in vitro and in vivo has been described (see, for example, International Patent Application Publication Nos. WO91 / 18088 and WO93 / 09239; U.S. Patent Nos. 4,797,368, 6,596,535, and 5,139,941, and European Patent No. 0488528, all of which are incorporated herein by reference in their entirety). These patent publications describe various AAV-derived constructs in which the rep and / or cap genes are deleted and replaced with genes of interest, and the use of these constructs to deliver genes of interest in vitro (into cultured cells) or in vivo (directly into an organism). Replication-deficient recombinant AAV can be prepared by co-transfecting a cell line infected with a human helper virus (e.g., adenovirus) with a plasmid containing the nucleic acid sequence of interest flanked by two AAV inverted terminal repeat (ITR) regions and a plasmid carrying the AAV encapsidation genes (rep and cap genes). The resulting AAV recombinant is then purified by standard techniques.

[0344] In some embodiments, the recombinant vector is encapsidated into a viral particle (e.g., an AAV viral particle including, but not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV16). Thus, the present disclosure includes recombinant viral particles (recombinant in that they contain a recombinant polynucleotide) containing any of the vectors described herein. Methods for producing such particles are known in the art and are described in U.S. Patent No. 6,596,535.

[0345] In some cases, the AAV vector can be modified to contain a modified VP protein (such as an AAV vector modified to contain a VP1 protein, a VP2 protein, or a VP3 protein). In one aspect, the AAV vector is a recombinant AAV (rAAV) vector. rAAV can be composed of a capsid sequence and structure substantially similar to that found in wild-type AAV (wtAAV). However, rAAV encapsulates a genome that is substantially free of AAV protein coding sequences and has a therapeutic gene expression cassette, such as a subject polynucleotide, designed in its place. In some cases, the viral-derived sequence can be an ITR, which may be required to direct genome replication and packaging during vector production. Suitable AAV vectors can be selected from any AAV serotype or a combination of serotypes. For example, the AAV vector can be any one of the following: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV 16. AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80 , AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.H In some cases, the AAV vector is selected based on its natural tropism. In some cases, the vector serotype is selected based on its ability to cross the blood-brain barrier. AAV9 and AAV10 have been shown to be able to cross the blood-brain barrier to transduce neurons and glial cells. In one aspect, the AAV vector is AAV2, AAV5, AAV6, AAV8 or AAV9. In some cases, the AAV vector is a chimera of at least two serotypes. In one aspect, the AAV vector is serotypes AAV2 and AAV5. In some cases, the chimeric AAV vector comprises rep and ITR sequences from AAV2 and a cap sequence from AAV5. In some cases, the chimeric AAV vector comprises rep and ITR sequences from AAV2 and a cap sequence from any other AAV serotype. In some embodiments, the AAV vector can be self-complementary. In some cases, the AAV vector can include inverted terminal repeats.In other cases, the AAV vector can include an inverted terminal repeat (scITR) sequence with a mutated terminal melting site. In some cases, the rep, cap, and ITR sequences from all the different AAV serotypes provided herein can be mixed and matched. In some cases, the AAV vector is from an adeno-associated virus having a serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV17, AAV18, AAV19, AAV20, AAV21, AAV22, AAV23, AAV24, AAV25, AAV26, AAV27, AAV28, AAV29, AAV30, AAV31, AAV32, AAV33, AAV34, AAV35, AAV36, AAV37, AAV38, AAV39, AAV40, AAV41, AAV42, AAV43, AAV44, AAV45, AAV46, AAV47, AAV48, AAV49, AAV50, AAV51, AAV52, AAV53, AAV54, AAV55, AAV56, AAV57, AAV58, AAV59, AAV60, AAV61, AAV62, AAV63, AAV64, AAV65, AAV66, AAV67, AAV68, AAV70, AAV71, AAV72, AAV73, AAV74, AAV75 16. AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc 80. AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, A AV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV .HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, AAV.HSC16 and AAVhu68. In some cases, the vector can be a recombinant AAV (rAAV) vector, a hybrid AAV vector, a chimeric AAV vector, a self-complementary AAV (scAAV) vector, a single-stranded AAV or any combination thereof. In some cases, the AAV vector includes a genome comprising a replication gene and an inverted terminal repeat from a first AAV serotype and a capsid protein from a second AAV serotype. In some cases, the AAV vector can be chimeric and can be: an AAV2 / 5 vector, an AAV2 / 6 vector, an AAV2 / 7 vector, an AAV2 / 8 vector or an AAV2 / 9 vector. In some cases, the inverted terminal repeat of the AAV vector includes a 5' inverted terminal repeat, a 3' inverted terminal repeat and a mutated inverted terminal repeat. In some cases, the mutated inverted terminal repeat lacks a terminal melting site. In some cases, suitable AAV vectors can be further modified to encompass modifications such as in the capsid or rep protein. Modifications can also include deletions, insertions, mutations and combinations thereof. In some cases, the vector is modified to reduce immunogenicity, thereby allowing repeated administration. In some cases, the serotype of the vector used is changed when repeated administration is performed to reduce and / or eliminate immunogenicity.

[0346] Pharmaceutical composition

[0347] Using the contents of the present disclosure, negative regulators that inhibit the genes or proteins of RCOR1, RCOR2, RCOR3, Sin3a, Sin3b, HDAC1, HDAC2, KDM1A, PHF21A, BAF53a, G9a, USP14, HuR, BrG1, EZH2, CDYL, or HMG20B can be screened out through various conventional screening methods.

[0348] In some embodiments, the inhibitor is one or more negative regulators that inhibit the expression of negatively regulated genes, or inhibit the mRNA level of negatively regulated genes, or inhibit the level of the encoded protein of negatively regulated genes.

[0349] Using the contents of the present disclosure, positive regulators that can enhance the gene expression level, mRNA level, protein expression level, or protein activity of DPYSL2, BAF45b, SCF, HuB, HuC, HuD, CYP1B1, or BTRC can be screened out through various conventional screening methods.

[0350] In some embodiments, the enhancer is one or more positive regulators that enhance the expression of positively regulated genes, or enhance the mRNA level of positively regulated genes, or enhance the level of the encoded protein of positively regulated genes.

[0351] The negative regulators or positive regulators that can be used in the present disclosure are substances that can reduce or eliminate the expression and / or activity of the gene, its RNA (such as mRNA) or its encoded protein at the DNA, RNA or protein level.

[0352] In some embodiments, the negative regulator includes an inhibitory antibody of a negatively regulated gene; or a small molecule inhibitor of a negatively regulated gene; or an inhibitory mRNA, microRNA, siRNA, shRNA, antisense oligonucleotide, binding protein or protein domain, polypeptide, nucleic acid aptamer, or PROTAC of a negatively regulated gene; or an inhibitory binding protein or ligand of a negatively regulated gene.

[0353] In some specific embodiments, the negative regulators of RCOR1 of the present disclosure include inhibitors that target a target sequence comprising SEQ ID NO: 68. The negative regulators of RCOR2 of the present disclosure include inhibitors that target a target sequence comprising SEQ ID NO: 69. The negative regulators of RCOR3 of the present disclosure include inhibitors that target a target sequence comprising SEQ ID NO: 70. The negative regulators of Sin3a of the present disclosure include inhibitors that target a target sequence comprising SEQ ID NO: 71. The negative regulators of Sin3b of the present disclosure include inhibitors that target a target sequence comprising SEQ ID NO: 72. The negative regulators of HDAC1 of the present disclosure include inhibitors that target a target sequence comprising SEQ ID NO: 73. The negative regulators of KDM1A of the present disclosure include inhibitors that target a target sequence comprising SEQ ID NO: 74. The negative regulators of HDAC2 of the present disclosure include inhibitors that target a target sequence comprising SEQ ID NO: 75. Negative regulators of PHF21A of the present disclosure include inhibitors targeting a target sequence comprising SEQ ID NO: 76. Negative regulators of BAF53a of the present disclosure include inhibitors targeting a target sequence comprising SEQ ID NO: 77. Negative regulators of G9a of the present disclosure include inhibitors targeting a target sequence comprising SEQ ID NO: 78. Negative regulators of USP14 of the present disclosure include inhibitors targeting a target sequence comprising SEQ ID NO: 79. Negative regulators of HuR of the present disclosure include inhibitors targeting a target sequence comprising SEQ ID NO: 80. Negative regulators of BrG1 of the present disclosure include inhibitors targeting a target sequence comprising SEQ ID NO: 81. Negative regulators of EZH2 of the present disclosure include inhibitors targeting a target sequence comprising SEQ ID NO: 82. Negative regulators of CDYL of the present disclosure include inhibitors targeting a target sequence comprising SEQ ID NO: 83. Negative regulators of HMG20B of the present disclosure include inhibitors that target a target sequence comprising SEQ ID NO:84.

[0354] In some embodiments, the negative regulators or positive regulators of the present disclosure act on astrocytes or MG cells.

[0355] In some embodiments, the positive regulator can be a substance that increases or enhances the expression and / or activity of a positively regulated gene, or its RNA (such as mRNA), or its encoded protein at the DNA, RNA, or protein level.

[0356] In some specific embodiments, positive regulators include: expression vectors containing promoters, endogenous expression activators, protein analogs or enhancers, epigenetic regulatory tools capable of increasing the expression of positive regulatory genes, positive regulatory gene expression activators, degradation inhibitors of proteins encoded by positive regulatory genes, stabilizers of positive regulatory gene mRNA, or exogenous positive regulatory genes or functional fragments of positive regulatory genes.

[0357] In a preferred embodiment, the method and steps of negative regulator regulation include neutralizing its protein using antibodies, silencing genes using shRNA or siRNA carried by viruses (such as adeno-associated viruses), or using gene editing tools.

[0358] The inhibition rate of a negative regulator on a negatively regulated gene is generally at least 50%, preferably at least 60%, 70%, 80%, 90%, or 95%. The inhibition rate can be controlled and detected using conventional techniques, such as flow cytometry, fluorescent quantitative PCR, or Western blot.

[0359] The negative regulators (including antibodies, antisense nucleic acids, gene editing tools and other inhibitors) or positive regulators (including: expression vectors containing promoters, endogenous expression activators, protein analogs or enhancers) of the present disclosure, when administered (administered) therapeutically, can inhibit or enhance the expression and / or activity of the gene or protein, thereby inducing glial cells to differentiate into neuronal cells, thereby treating diseases associated with neuronal dysfunction or death. Typically, these substances can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous medium, wherein the pH is generally about 5-8, preferably about 6-8, although the pH value may vary with the properties of the formulated substance and the condition to be treated. The formulated pharmaceutical composition can be administered by conventional routes, including (but not limited to): topical, intramuscular, intracranial, intraocular, intraperitoneal, intravenous, subcutaneous, intradermal, topical administration, autologous cell extraction and culture followed by reinfusion, etc.

[0360] The present disclosure also provides a pharmaceutical composition containing a safe and effective amount of a negative regulator or positive regulator of the present disclosure and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should be compatible with the mode of administration. The pharmaceutical composition of the present disclosure can be prepared in the form of an injection, for example, by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as tablets and capsules can be prepared by conventional methods. Pharmaceutical compositions such as injections, solutions, tablets and capsules are preferably manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 1 microgram to 10 mg / kg body weight per day.

[0361] Key advantages of this disclosure include:

[0362] (1) The present disclosure discovers for the first time that reducing the expression or activity of RCOR1, RCOR2, RCOR3, Sin3a, Sin3b, HDAC1, HDAC2, KDM1A, PHF21A, BAF53a, G9a, USP14, HuR, BrG1, EZH2, CDYL, or HMG20B genes or their encoded proteins in non-neuronal cells, particularly glial cells, and / or increasing the expression or activity of DPYSL2, BAF45b, SCF, HuB, HuC, HuD, CYP1B1, or BTRC genes or their encoded proteins in glial cells can induce the differentiation of glial cells into dopamine neurons, thereby preventing and / or treating diseases associated with neuronal dysfunction or death.

[0363] (2) The negative regulator or positive regulator provided by the present invention can induce the generation of dopamine neurons, thereby having a potential effect in treating Parkinson's disease.

[0364] (3) The present disclosure directly converts Muller cells into RGC cells or photoreceptor cells by inhibiting the expression of RCOR1, RCOR2, RCOR3, Sin3a, Sin3b, HDAC1, HDAC2, KDM1A, PHF21A, BAF53a, G9a, USP14, HuR, BrG1, EZH2, CDYL, or HMG20B in the retina, and / or enhancing the expression of DPYSL2, BAF45b, SCF, HuB, HuC, HuD, CYP1B1, or BTRC in glial cells.

[0365] (4) Regenerated RGCs can be integrated into the visual pathway and improve visual function in RGC injury mouse models.

[0366] The present disclosure is further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present disclosure only and are not intended to limit the scope of the present disclosure. The experimental methods in the following examples, for which specific conditions are not specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight.

[0367] Unless otherwise specified, the materials and reagents used in the examples of this disclosure are commercially available products.

[0368] General Methods

[0369] Animal ethics: The use and maintenance of animals complied with the guidelines of the Biomedical Research Ethics Committee of the Center for Excellence in Brain Science and Intelligence Technology, Chinese Academy of Sciences.

[0370] Construction of guide RNA sequence and AAV expression plasmid: In order to overexpress nSR100 in glial cells, the glial cell-specific promoter GFAP was used to promote the expression of nSR100.

[0371] Transient transfection of astrocytes and qPCR: Primary mouse astrocytes were isolated and seeded in 6-well plates. 3 μg of a vector expressing gRNA-CasRx-GFP was transiently transfected using Lipofectamine 3000 (Thermo Fisher Scientific) according to standard procedures. A control plasmid expressed a non-targeting gRNA. One to two days after transient transfection, GFP-positive cells were collected by flow cytometry cell sorting (FACS) and lysed for qPCR analysis. The qPCR analysis method was as follows: RNA was extracted using Trizol (Ambion), and then reverse transcribed into cDNA using a reverse transcription kit (HiScript Q RT SuperMix for qPCR, Vazyme, Biotech). Amplification was tracked using AceQ qPCR SYBR Green Master Mix (Vazyme, Biotech). qPCR primers are shown in Table 2 below:

[0372] Table 2: qPCR primers

[0373] Targeted injection: AAV8 was used to carry the target gene in this study. The titer of AAV-CasRx, AAV-CasRx-gRNA, or AAV-GFAP-positive regulatory gene was greater than 5×10 12 vg / ml (1-3 μl per injection). AAV was injected into the striatum (AP +0.8 mm, ML ±1.6 mm and DV -2.8 mm).

[0374] Immunofluorescence staining: 6-12 weeks after injection, mice were perfused, and brains were removed and fixed overnight with 4% paraformaldehyde (PFA) and dehydrated in 30% sucrose for at least 12 hours. After embedding, sections were frozen and sliced ​​at a thickness of 30 μm. Before immunofluorescence staining, brain sections were thoroughly rinsed with 0.1 M phosphate buffered saline (PBS). Primary antibodies: rabbit polyclonal NeuN antibody (Brain, 1:500, #ABN78, Millipore), mouse TH antibody (1:300, MAB318, Millipore). Secondary antibodies: donkey anti-mouse (#715-545-150, Jackson ImmunoResearch) and donkey anti-rabbit (#711-545-152, Jackson ImmunoResearch) were used in this study. After antibody incubation, sections were washed and covered with mounting medium (Life Technology).

[0375] Statistical analysis: Error bars were set by SEM, and statistical significance (p < 0.05) was calculated using unpaired two-tailed t-test or one-way analysis of variance. All experiments were randomized, and no statistical method was used to predetermine the sample size.

[0376] For RNA-seq, 293T or N2a cells were cultured in 15-cm dishes and transiently transfected with 70 μg of plasmid. The top 20% of GFP- and mCherry-positive N2a cells were collected by FACS, and RNA was extracted and converted to cDNA for whole-transcriptome RNA-seq.

[0377] Intravitreal and subretinal injections: NMDA and AAV8 were introduced by intravitreal and subretinal injections, respectively, as described previously.

[0378] Subretinal injection: AAV (>1×10 13vg / ml). To determine reprogramming in the intact retina, a total of 1 μl of GFAP-GFP-Cre (0.2 μl) and AAV-GFAP-nSR100, or GFAP-CasRx-gRNA (0.8 μl), or GFAP-GFP-Cre (0.2 μl) and GFAP-CasRx (0.8 μl) was delivered to the retina by subretinal injection (Ai9 and C57BL / 6 mice, 5 weeks to 12 months of age). To determine reprogramming in the damaged retina, NMDA was dissolved in PBS to a concentration of 200 mM, and then 1.5 μl of NMDA solution was injected into the eyes of 4-8 week-old Ai9 mice or 5-6 week-old C57BL / 6 mice by intravitreal injection (for VEP and black-white scene preference test). 2-3 weeks after NMDA injection, each virus combination was co-delivered to the retina by subretinal injection.

[0379] Immunofluorescence staining: 1.5-3 months after AAV injection, eyes, optic nerves, and brains were harvested and fixed with 4% paraformaldehyde (PFA) for 2 hours (eyes and optic nerves) or 24 hours (brains), followed by dehydration in 30% sucrose solution for 2 hours (eyes and optic nerves) or 24 hours (brains). After embedding and freezing, eyes and brains were sectioned at 30 μm thickness. Primary antibodies used for immunofluorescence staining included rabbit anti-RBPMS (1:500, 15187-1-AP, Proteintech), and secondary antibodies included Jackson ImmunoResearch and Cy™ 5 AffiniPure Donkey rabbit anti-IgG (H+L) (1:500, 711-175-152, Jackson ImmunoResearch). After antibody incubation, slides were washed with PBS and mounted with anti-quencher. Images were acquired using an Olympus FV3000 microscope.

[0380] Example

[0381] Example 1: In vitro specific knockdown using CasRx

[0382] To evaluate the knockdown of negatively regulated genes, gRNAs targeting RCOR1, Sin3a, HDAC2, KDM1A, PHF21A, BAF53a, G9a, USP14, HuR, BrG1, EZH2, CDYL, and HMG20B were designed, and CasRx and gRNA were co-transfected into N2A cells.

[0383] Among them, the nucleotide sequence of gRNA and target sequence are shown in Table 1 above.

[0384] Transfections were performed by transiently transfecting 4 μg of the CAG-CasRx-P2A-GFP plasmid and 2 μg of the U6-gRNA-CMV-mCherry plasmid to determine the inhibitory effect of each gRNA in the cell line in vitro. Single transfections with the CAG-CasRx-P2A-GFP plasmid served as a control. Lipofectamine 3000 (Thermo Fisher Scientific) was used according to standard protocols. Two days after transfection, 30,000 GFP- and mCherry-positive cells were collected from each sample by fluorescence-activated cell sorting (FACS), lysed, and analyzed by qPCR. qPCR primers are listed in Table 2.

[0385] The results are shown in Figure 1. Co-transfection of gRNA targeting the mRNA of the negative regulatory gene and CasRx can achieve significant knockdown of the target gene in mouse N2A cells.

[0386] Example 2: Study on the effect of negative regulatory genes on the differentiation of astrocytes into neurons

[0387] To investigate whether knockdown of negative regulatory genes could induce astrocyte-to-neuronal or dopaminergic neuronal transdifferentiation, C57BL / 6 mice older than eight weeks were injected with AAV-GFAP-CasRx-gRNA or the control vectors AAV-GFAP-CasRx and AAV-GFAP-mCherry, respectively, into the striatum. Plasmid designs are shown in Figures 2A and 2A. Either vector 1 plus vector 2 or vector 1 plus vector 3 were injected. GFAP is an astrocyte-specific promoter, AAV-GFAP-mCherry specifically labels astrocytes, and AAV-GFAP-CasRx serves as the control AAV. The gRNAs target the negative regulatory genes RCOR1, HDAC2, PHF21A, BAF53a, G9a, USP14, HuR, BrG1, EZH2, CDYL, HMG20B, and KDM1A, respectively.

[0388] Samples were collected and analyzed 1-3 months after injection. The results are shown in Figure 3. As shown in Figure 3A, in the control group injected with GFAP-mCherry + AAV-GFAP-CasRx, mCherry-positive red cells showed typical glial cell morphology, and no red cells with obvious neuronal morphology were found. Further immunofluorescence staining with the neuron-specific marker protein NeuN revealed that almost no red cells in the control group were co-labeled with NeuN, indicating that GFAP-mCherry can specifically label astrocytes and that the control group injected with GFAP-mCherry + AAV-GFAP-CasRx cannot induce astrocyte transdifferentiation into neurons. As shown in Figures 3B to 3M , in the experimental groups injected with GFAP-mCherry+AAV-GFAP-CasRx-gRNA, different numbers of mCherry-positive cells were co-labeled with NeuN, indicating that RCOR1, HDAC2, PHF21A, BAF53a, G9a, USP14, HuR, BrG1, EZH2, CDYL, HMG20B, and KDM1A can induce the transdifferentiation of glial cells into neurons to varying degrees.

[0389] As shown in Figure 4, after knocking down different negative regulatory genes, astrocytes were converted into neurons. Among them, RCOR1, G9a, BAF53A and others had a higher efficiency in inducing the transdifferentiation of glial cells into neurons.

[0390] Example 3: Study on the effect of positive regulatory genes on the transdifferentiation of astrocytes into neurons

[0391] In order to study the effect of overexpression of positive regulatory genes on transdifferentiation, attempts were made to overexpress DPYSL2, BAF45b, SCF, HuB, HuC, HuD, CYP1B1, and BTRC in the mouse striatum.

[0392] Mice were injected with either a control or test AAV vector into the striatum or substantia nigra. Vector 1 contained AAV-GFAP-mCherry, while vector 2 (AAV-GFAP-Gene) drove target gene expression via the astrocyte-specific GFAP promoter. Similar to the target gene knockdown groups, samples were collected 1-3 months after AAV injection to analyze the efficiency of glial cell-to-neuron transdifferentiation induced by each target. The results are shown in Figures 5 and 6.

[0393] As shown in Figure 5A, in the brain slices of mice injected with the AAV-GFAP-mCherry control group AAV, the red fluorescent labeled cells were typical astrocyte morphology, and there were almost no cells co-labeled with mCherry and NeuN. In the test group AAV injection (AAV-GFAP-mCherry + AAV-GFAP-positive regulatory gene), in which the positive regulatory genes were DPYSL2, BAF45b, SCF, HuB, HuC, HuD, CYP1B1, and BTRC, there were different proportions of cells co-labeled with mCherry and NeuN (Figures 5B to 5I). These results show that overexpression of genes such as DPYSL2, BAF45b, SCF, HuB, HuC, HuD, CYP1B1, and BTRC can induce the transdifferentiation of astrocytes to neurons to varying degrees. As shown in Figure 6, SCF, HuB, HuC, and CYP1B1 all showed excellent transdifferentiation efficiency.

[0394] Example 4: Transdifferentiation of astrocytes into dopamine neurons

[0395] To further investigate whether overexpression of positive regulatory genes and inhibition of negative regulatory genes can promote the transdifferentiation of astrocytes into dopamine neurons, referring to the procedures of Examples 1 to 3, we injected AAV of the control group or the test group into the striatum of mice and stained them with the dopamine neuron-specific protein marker TH. The results are shown in Figure 7. No TH-positive cells were found in the control group (Figure 7A), while TH-positive dopamine neurons co-labeled with mCherry signals were found in the HuB and BTRC overexpression groups, indicating that overexpression of HuB and BTRC can induce the transdifferentiation of astrocytes into dopamine neurons.

[0396] Example 5: Transdifferentiation of Muller glial cells into photoreceptor cells

[0397] In the retina, Müller glia perform functions similar to those of glial cells, and the retina contains neurons with a variety of specialized functions, such as photoreceptors and bipolar cells. In order to explore whether each gene can transdifferentiate Müller glial cells into photoreceptor cells in the retinal system, we used the GFAP-EGFP-2A-Cre system in combination with Ai9 mice to achieve specific labeling of Müller glial cells. The operation diagram is shown in Figure 8. Referring to the operations of Examples 1 to 3, a mixed virus of AAV-GFAP-CasRx-gRNA (or control vector AAV-GFAP-CasRx) and AAV-GFAP-EGFP-2A-Cre was injected subretinaly into Ai9 (Rosa26-CAG-LSL-tdTomato-WPRE) mice aged 6-10 weeks. AAV-GFAP-EGFP-2A-Cre is used to specifically mark Müller glial cells or neurons converted from glial cells (retinal ganglion cells or photoreceptor cells). After 1-3 months of injection, the retina was sliced ​​and analyzed to study whether the corresponding negative regulatory genes were inhibited or the corresponding positive regulatory genes were upregulated to see whether the retinal system can transdifferentiate Müller glial cells into photoreceptor cells.

[0398] GFAP-EGFP-2A-Cre+GFAP-CasRx was used as the control group, and the group expressing GFAP-EGFP-2A-Cre+GFAP-CasRx-gRNA was used as the test group, where the gRNA targeted Sin3a, HDAC2, BAF53a, G9a, HuR, BrG1, EZH2, CDYL, and KDM1A, respectively.

[0399] GFAP-EGFP-2A-Cre was used as the control group, and GFAP-EGFP-2A-Cre+GFAP-positive regulatory genes were used as the test group, where the positive regulatory genes were DPYSL2, BAF45b, SCF, CYP1B1, and BTRC genes.

[0400] Different groups of AAV were injected into the subretina of Ai9 mice with NMDA model and analyzed 1-3 months after injection.

[0401] The effect of negative regulatory gene groups on transdifferentiation is shown in Figure 9. In Figure 9A, in the GFAP-EGFP-2A-Cre group, Müller glial cells showed typical morphology, with cell bodies located in the inner granular layer (INL) and processes distributed to the outer granular layer (ONL) and retinal ganglion cell layer (GCL), respectively. No red signal-labeled photoreceptor cells were observed in the ONL, indicating that no Müller glial cells transdifferentiated into photoreceptor cells in the control group. As shown in Figures 9B-9J, in the experimental group with negative regulatory gene knockdown, a large number of red fluorescent-labeled photoreceptor cells were found in the Sin3a, BAF53a, G9a, HuR, BrG1, EZH2, and KDM1A groups. These results indicate that knocking down Sin3a, HDAC2, BAF53a, G9a, HuR, BrG1, EZH2, CDYL, and KDM1A genes can transdifferentiate Müller glial cells into photoreceptor cells.

[0402] The effect of positively regulated gene sets on transdifferentiation is shown in Figure 10 . Red fluorescent-labeled photoreceptor cells were found in the ONL layer of BAF45b and DPYSL2, indicating that overexpression of DPYSL2 and BAF45b can induce transdifferentiation of Müller glial cells into photoreceptor cells.

[0403] In the retina, the number of Müller glial cells reprogrammed into photoreceptor cells by knocking down the expression of negative regulatory genes or overexpressing positive regulatory genes by CasRx is shown in Figure 11, which shows that inhibition of Sin3a, KDM1A, BAF53a, G9a, HuR, BrG1, EZH2, and CDYL all exhibited excellent photoreceptor cell regeneration ability, inhibition of HDAC2 also had photoreceptor cell regeneration ability, and overexpression of DPYSL2 and BAF45b also had photoreceptor cell regeneration ability.

[0404] Photoreceptor cells are divided into cones and rods. We selected the Sin3a and KDM1A groups and stained them with rod-specific protein markers. As shown in Figure 12, we found that most of the photoreceptor cells that were transformed from Müller glial cells in the ONL layer were rods.

[0405] We stained some groups with the cone-specific protein marker m-CAR and performed imaging analysis under a confocal microscope. The results are shown in Figure 13, showing the presence of cones.

[0406] Example 6: Transdifferentiation of Müller glial cells into optic ganglion cells

[0407] To investigate whether knockdown of RCOR1, Sin3a, HDAC2, KDM1A, PHF21A, BAF53a, G9a, USP14, HuR, BrG1, EZH2, CDYL, HMG20B, or overexpression of DPYSL2, BAF53b, BAF45b, SCF, HuB, HuC, HuD, CYP1B1, or BTRC could induce the transdifferentiation of Müller glia into retinal ganglion cells (RGCs), we subretinally injected various AAV combinations into 6-8-week-old Ai9 (Rosa26-CAG-LSL-tdTomato-WPRE) mice induced by NMDA. In the gene knockdown group, we injected a mixture of AAV-GFAP-CasRx-gRNA (or the control vector AAV-GFAP-CasRx) and AAV-GFAP-EGFP-2A-Cre. AAV-GFAP-EGFP-2A-Cre is used to specifically mark Müller glial cells or neurons (optic ganglion cells) converted from glial cells. In the overexpression group, AAV-GFAP-EGFP-2A-Cre was injected as a control group, and AAV-GFAP-EGFP-2A-Cre + AAV-GFAP-positive regulatory genes (wherein the positive regulatory genes are DPYSL2, BAF45b, SCF, HuB, HuC, HuD, CYP1B1, BTRC) were injected. Because the pseudopodia of Müller glial cells in the GCL layer significantly interfere with the observation of transdifferentiated RGC cells, and each RGC cell sends an axon to the optic nerve, we used optic nerve imaging analysis methods and analyzed the results 1-3 months after injection. The results are shown in Figures 14 and 15. As shown in Figure 14A, in the control group, we found very few optic nerves, with an average of less than 5 axons labeled with red fluorescent signals per optic nerve. As shown in Figures 14B to 14I, in the HuR knockdown group, a large number of axons labeled with red fluorescent signals were observed, while in the KDM1A, Sin3a, and HDAC2 groups, almost no obvious axonal signals labeled with red fluorescent signals were observed. As shown in Figure 15, in the SCF, HuD, and CYP1B1 overexpression groups, a large number of axons labeled with red fluorescent signals were observed, with the SCF group having the most axons labeled with red fluorescent signals. These results indicate that knocking down HuR expression or overexpressing SCF, HuD, and CYP1B1 can promote the transdifferentiation of Müller glia into RGCs, while genes such as KDM1A, Sin3a, and HDAC2 cannot promote the transdifferentiation of Müller glia into RGCs.

[0408] Knockdown of KDM1A, HDAC2, BAF53a, G9a, HuR, BrG1, EZH2, and CDYL expression or overexpression of DPYSL2, BAF45b, SCF, HuB, HuD, CYP1B1, and BTRC in the retina reprogrammed Müller glial cells into retinal ganglion cells. The statistical graph of the number of tdTomato-positive axons in the optic nerve is shown in Figure 16. More retinal ganglion cells were found in the SCF, HuD, and CYP1B1 groups.

[0409] Example 7: Photoreceptor cell regeneration was found in RP disease model mice

[0410] Methods: The Pde6b gene is a pathogenic gene associated with retinitis pigmentosa (RP). Defects in the Pde6b gene lead to the death of photoreceptor cells. Currently, Pde6b-deficient mice have been shown to be an animal model of the disease with the genetic characteristics of RP. This patent utilizes a Pde6b knockout mouse model constructed using CRISPR-Cas9 editing. Immunohistochemistry demonstrates that Pde6b knockout mice experience massive rod cell death at 4 weeks of age, and complete rod cell death by 6 weeks (Figures 17A-C). At 7 weeks of age, the disease model mice were subretinally injected with AAVs targeting KDM1A and EZH2 (AAV-U6-gRNA (KDM1A)-GFAP-CasRx and AAV-U6-gRNA (EZH2)-GFAP-CasRx) and AAV overexpressing Pde6b (AAV-CBH-Pde6b). The control group was injected with control AAV (AAV-GFAP-CasRx) and AAV overexpressing Pde6b (AAV-CBH-Pde6b). The injection volume of the transdifferentiation target AAV was 1x10 10 vg, Pde6b overexpression AAV injection volume was 5x10 9vg, the total volume of AAV injected was 1 μl. Two months after injection, retinal tissue was taken for analysis, and it was found that there were no photoreceptors in the outer granular layer of the retina of mice injected with the control group AVV (AAV-GFAP-CasRx+AAV-CBH-Pde6b). No Rhodopsin-positive cells were found when stained with the rod cell-specific marker Rhodopsin (Figure 17D). In the group injected with AAV (AAV-U6-gRNA (KDM1A)-GFAP-CasRx and AAV-U6-gRNA (EZH2)-GFAP-CasRx) that targeted the transdifferentiation targets KDM1A and EZH2, a layer of cells was found in the outer granular layer of the retina of mice. Immunofluorescence staining showed that these cells were Rhodopsin-positive photoreceptors (Figure 17E-F). Statistical analysis of each group revealed that in the KDM1A and EZH2 knockdown groups, approximately 20 Rhodopsin-positive photoreceptor cells were generated within 200 μm (Figure 17G). Analysis of the thickness of the outer granular layer revealed that the outer granular layer was significantly thickened in the KDM1A and EZH2 knockdown groups (Figure 17H).

[0411] In the above-mentioned embodiments (such as Example 3 and Example 4), the applicant describes in detail the results of injecting the negative modulator or positive modulator of the present application into the striatum. Those skilled in the art can choose to perform targeted injections in other parts of the brain based on the technical solution of the present application and the common knowledge in the field, such as injections into the ventral tegmental area of ​​the midbrain, spinal cord, hypothalamus, dorsal midbrain, cerebral cortex, hippocampus, cerebellum, or substantia nigra, etc., especially injections into the substantia nigra, which also have the same or similar technical effects.

[0412] In the above-mentioned embodiments (e.g., Example 5), the applicant described in detail the results of subretinal injection of the negative modulator or positive modulator of the present application. Based on the technical solution of the present application and the common knowledge in the field, those skilled in the art may choose to perform targeted injection in other parts of the eye, such as the vitreous cavity, which also has the same or similar technical effects.

[0413] It should be understood that the above embodiments are merely illustrative and not specific limitations. For example, in the above embodiments, the applicant describes in detail the results of negatively regulating the RCOR1 gene. Those skilled in the art can select genes similar to RCOR1 (such as RCOR2 or RCOR3) for regulation based on the technical solution of the present application and the general knowledge in the art, which also have the same or similar technical effects; the applicant describes in detail the results of negatively regulating the Sin3a gene. Those skilled in the art can select genes similar to Sin3a (such as Sin3b) for regulation based on the technical solution of the present application and the general knowledge in the art, which also have the same or similar technical effects; the applicant describes in detail the results of negatively regulating the HDAC2 gene. Those skilled in the art can select genes similar to HDAC2 (such as HDAC1) for regulation based on the technical solution of the present application and the general knowledge in the art, which also have the same or similar technical effects.

[0414] All documents mentioned in this disclosure are incorporated herein by reference, just as if each document were individually incorporated by reference. It should also be understood that after reading the above teachings of this disclosure, those skilled in the art may make various changes or modifications to this disclosure, and that such equivalents also fall within the scope of the claims appended hereto.

[0415] The following is the sequence information of the positive and negative regulatory genes involved in this application:

[0416] Human RCOR1 protein sequence (SEQ ID NO: 1)

[0417] Human RCOR1 coding sequence (SEQ ID NO: 2)

[0418] Human RCOR2 protein sequence (SEQ ID NO: 3)

[0419] Human RCOR2 coding sequence (SEQ ID NO: 4)

[0420] Human RCOR3 protein sequence (SEQ ID NO: 5)

[0421] Human RCOR3 coding sequence (SEQ ID NO: 6)

[0422] Human Sin3a protein sequence (SEQ ID NO: 7)

[0423] Human Sin3a coding sequence (SEQ ID NO: 8)

[0424] Human Sin3b protein sequence (SEQ ID NO: 9)

[0425] Human Sin3b coding sequence (SEQ ID NO: 10)

[0426] Human HDAC1 protein sequence (SEQ ID NO: 11)

[0427] Human HDAC1 coding sequence (SEQ ID NO: 12)

[0428] Human HDAC2 protein sequence (SEQ ID NO: 13)

[0429] Human HDAC2 coding sequence (SEQ ID NO: 14)

[0430] Human Kdm1a protein sequence (SEQ ID NO: 15)

[0431] Human Kdm1a coding sequence (SEQ ID NO: 16)

[0432] Human PHF21A protein sequence (SEQ ID NO: 17)

[0433] Human PHF21A coding sequence (SEQ ID NO: 18)

[0434] Human BAF53a protein sequence (SEQ ID NO: 19)

[0435] Human BAF53a coding sequence (SEQ ID NO: 20)

[0436] Human G9a protein sequence (SEQ ID NO: 21)

[0437] Human G9a coding sequence (SEQ ID NO: 22)

[0438] Human USP14 protein sequence (SEQ ID NO: 23)

[0439] Human USP14 coding sequence (SEQ ID NO: 24)

[0440] Human HuR protein sequence (SEQ ID NO: 25)

[0441] Human HuR coding sequence (SEQ ID NO: 26)

[0442] Human BrG1 protein sequence (SEQ ID NO: 27)

[0443] Human BrG1 coding sequence (SEQ ID NO: 28)

[0444] Human EZH2 protein sequence (SEQ ID NO: 29)

[0445] Human EZH2 coding sequence (SEQ ID NO: 30)

[0446] Human CDYL protein sequence (SEQ ID NO: 31)

[0447] Human CDYL coding sequence (SEQ ID NO: 32)

[0448] Human HMG20B protein sequence (SEQ ID NO: 33)

[0449] Human HMG20B coding sequence (SEQ ID NO: 34)

[0450] Human DPYSL2 protein sequence (SEQ ID NO: 35)

[0451] Human DPYSL2 coding sequence (SEQ ID NO: 36)

[0452] Human BAF45b protein sequence (SEQ ID NO: 37)

[0453] Human BAF45b coding sequence (SEQ ID NO: 38)

[0454] Human SCF protein sequence (SEQ ID NO: 39)

[0455] Human SCF coding sequence (SEQ ID NO: 40)

[0456] Human HuB protein sequence (SEQ ID NO:41)

[0457] Human HuB coding sequence (SEQ ID NO: 42)

[0458] Human HuC protein sequence (SEQ ID NO: 43)

[0459] Human HuC coding sequence (SEQ ID NO: 44)

[0460] Human HuD protein sequence (SEQ ID NO: 45)

[0461] Human HuD coding sequence (SEQ ID NO: 46)

[0462] Human CYP1B1 protein sequence (SEQ ID NO: 47)

[0463] Human CYP1B1 coding sequence (SEQ ID NO: 48)

[0464] Human BTRC protein sequence (SEQ ID NO: 49)

[0465] Human BTRC coding sequence (SEQ ID NO: 50)

Claims

1. A method for transdifferentiating non-neuronal cells of a mammal into neurons, wherein: include: Provided are negative regulators capable of reducing the expression of negative regulatory genes, including RCOR1, RCOR2, RCOR3, Sin3a, Sin3b, HDAC1, HDAC2, KDM1A, PHF21A, BAF53a, G9a, USP14, HuR, BrG1, EZH2, CDYL, or HMG20B, and / or Providing positive regulators capable of increasing the expression of positively regulated genes, wherein the positively regulated genes include DPYSL2, BAF45b, SCF, HuB, HuC, HuD, CYP1B1, or BTRC, An effective amount of the negative regulator or the positive regulator contacts the non-neuronal cells to induce the non-neuronal animal cells to transdifferentiate into neurons.

2. The method according to claim 1, wherein The non-neuronal cells are of human origin.

3. The method according to claim 1 or 2, wherein The non-neuronal cells are stem cells, progenitor cells or terminally differentiated cells; preferably, they are glial cells; more preferably, the glial cells are astrocytes, oligodendrocytes, microglia, NG2 cells, Muller glial cells, glioma cells or spiral ganglion glial cells, and more preferably, the glial cells are astrocytes or Muller glial cells.

4. The method according to claim 3, wherein: The non-neuronal cells are derived from the brain. Preferably, the non-neuronal cells are derived from the cerebrum, midbrain, cerebellum, brainstem, and spinal cord; more preferably, they are derived from the striatum or substantia nigra in the brain.

5. The method according to claim 4, wherein: The non-neuronal cells are astrocytes derived from the brain, preferably, astrocytes derived from the striatum or substantia nigra.

6. The method of claim 4, wherein: The non-neuronal cells are derived from the eye. Preferably, the non-neuronal cells are Muller glial cells from the eye.

7. The method according to any one of claims 1 to 6, wherein: The neuron is a dopaminergic neuron, a retinal ganglion cell, a photoreceptor cell, a 5-HT neuron, a NE neuron, a ChAT neuron, a motor neuron, a GABA neuron, a glutamatergic neuron, a spinal cord neuron, a spinal cord motor neuron, a spinal cord sensory neuron, a bipolar cell, an amacrine cell, a cochlear neuron, a pyramidal neuron, an interneuron, a medium spiny neuron, a Purkinje cell, a granule cell, an olfactory sensory neuron, or a periglomerular cell, or a combination thereof.

8. The method according to any one of claims 1 to 7, wherein: The negative regulatory gene is RCOR1, RCOR2, RCOR3, Sin3a, Sin3b, KDM1A, BAF53a, G9a, HuR, BrG1, or EZH2, and the non-neuronal animal cells are derived from the brain or eyes.

9. The method of claim 8, wherein: The negative regulatory gene is RCOR1, RCOR2, RCOR3 or G9a, and the non-neuronal animal cells are derived from the brain; preferably, the non-neuronal cells are astrocytes from the brain; more preferably, the non-neuronal cells are astrocytes from the striatum or substantia nigra.

10. The method of claim 8, wherein: The negative regulatory genes are Sin3a, Sin3b, KDM1A, BAF53a, G9a, HuR, BrG1, CDYL, or EZH2, and the non-neuronal cells are derived from the eyes; preferably, the non-neuronal cells are Muller glial cells from the eyes.

11. The method according to claim 10, wherein: The negative regulatory gene is HuR, and the neurons are retinal ganglion cells.

12. The method of claim 10, wherein: The negative regulatory gene is Sin3a, KDM1A, BAF53a, G9a, HuR, BrG1, CDYL, or EZH2, and the neuron is a photoreceptor cell.

13. The method according to any one of claims 1 to 7, wherein: The positively regulated genes are DPYSL2, BAF45b, SCF, HuB, HuC, HuD, CYP1B1, or BTRC, and the non-neuronal cells are from the brain; preferably, the non-neuronal cells are from the striatum, substantia nigra, ventral tegmental area of ​​the midbrain, spinal cord, hypothalamus, dorsal midbrain, cerebral cortex, hippocampus, and cerebellum; more preferably, they are from the striatum.

14. The method of claim 13, wherein: The positive regulatory gene is SCF or HuB.

15. The method of claim 13, wherein: The positive regulatory gene is HuB or BTRC, and an effective amount of the positive regulator contacts the non-neuronal cells to induce the non-neuronal cells to transdifferentiate into dopaminergic neurons.

16. The method according to any one of claims 13 to 15, wherein: The non-neuronal cells are astrocytes; more preferably, the non-neuronal cells are astrocytes from the striatum or substantia nigra.

17. The method according to any one of claims 1 to 7, wherein: The positively regulated genes are DPYSL2, BAF45b, SCF, HuC, HuD, or CYP1B1, and the non-neuronal cells are from the eyes; preferably, the non-neuronal cells are Muller glial cells from the eyes.

18. The method of claim 17, wherein: The positive regulatory gene is SCF, HuD, or CYP1B1, and the neuron is a retinal ganglion cell.

19. The method of claim 17, wherein: The positively regulated gene is DPYSL2 or BAF45b, and the neurons are photoreceptor cells.

20. The method according to any one of claims 1 to 19, wherein: The reducing of negative regulatory gene expression is to reduce the gene level of the negative regulatory gene, or reduce the mRNA level of the negative regulatory gene, or reduce the expression level of the protein encoded by the negative regulatory gene; The improvement of the positive regulatory gene expression is to increase the gene level of the positive regulatory gene, or to increase the mRNA level of the positive regulatory gene, or to increase the expression level of the protein encoded by the positive regulatory gene.

21. The method of claim 20, wherein: The negative regulator is selected from gene editing tools or epigenetic regulation tools that reduce the expression of negative regulatory genes; inhibitors of negative regulatory genes, inhibitors of negative regulatory gene activity, or degradation activators of the encoded proteins of negative regulatory genes.

22. The method of claim 21, wherein: The inhibitor is: an inhibitory antibody of a negative regulatory gene; or a small molecule inhibitor of a negative regulatory gene; or an inhibitory mRNA, microRNA, siRNA, shRNA, antisense oligonucleotide, binding protein or protein domain, polypeptide, nucleic acid aptamer, or PROTAC of a negative regulatory gene; or an inhibitory binding protein or ligand of a negative regulatory gene.

23. The method of claim 21, wherein: The negative regulator contains the gRNA and gene editing protein as described in any one of SEQ ID NOs: 51-67.

24. The method of claim 20, wherein: The positive regulator is selected from epigenetic regulatory tools capable of increasing the expression of positive regulatory genes, positive regulatory gene expression activators, degradation inhibitors of positive regulatory gene encoded proteins, stabilizers of positive regulatory gene mRNA, or exogenous positive regulatory genes or functional fragments of positive regulatory genes.

25. The method of claim 24, wherein: The activator is: an agonistic antibody of a positively regulated gene; or a small molecule agonist of a positively regulated gene; or an agonistic binding protein or ligand of a positively regulated gene; or an inhibitor of a competitive gene of a positively regulated gene.

26. The method of claim 24, wherein: The positive regulator contains a nucleic acid sequence as shown in SEQ ID NO: 36, 38, 40, 42, 44, 46, 48, or 50, or a functional fragment of a nucleic acid sequence as shown in SEQ ID NO: 36, 38, 40, 42, 44, 46, 48, or 50.

27. The gene editing tool according to claim 21, wherein The gene editing tool includes a gene editing system or an expression vector thereof, wherein the gene editing system is selected from: CRISPR system (including CRISPR / Cas system), ZFN system, TALEN system, or a combination thereof 28. The method according to claim 27, wherein The CRISPR system is used to reduce the expression or activity of negative regulatory genes; preferably, the CRISPR system contains a nucleic acid encoding a Cas enzyme or a functional domain of a Cas enzyme and a gRNA targeting the cell transdifferentiation factor; more preferably, the Cas enzyme is Cas13; more preferably, the Cas enzyme is Cas13d, Cas13X, Cas13a, Cas13b, Cas13c, or Cas13Y; more preferably, the Cas enzyme is CasRx.

29. The method according to claim 20, wherein The negative regulator or positive regulator is carried by a carrier; preferably, the carrier is a viral vector, lipid nanoparticles (LNP), liposomes, cationic polymers (such as PEI), nanoparticles, exosomes, or virus-like particles; more preferably, the carrier is an AAV vector or lipid nanoparticles.

30. The method according to any one of claims 1 to 29, wherein: The effective amount of the negative regulator or the positive regulator is contacted with the non-neuronal cells in vitro to induce the non-neuronal cells to transdifferentiate into neurons in vitro; or The effective amount of the negative regulator or the positive regulator contacts the non-neuronal cells in vivo to induce the non-neuronal cells to transdifferentiate into neurons in vitro.

31. Use of the negative regulator or positive regulator according to any one of claims 1 to 30 for preparing a medicament for preventing or treating diseases associated with neuronal damage or neuronal death.

32. Use of the negative regulator or positive regulator according to any one of claims 1 to 30 for preventing or treating diseases associated with neuronal damage or neuronal death.

33. The use according to claim 31 or 32, wherein The drug is formulated for in vivo administration to the nervous, visual, and auditory systems.

34. The use according to claim 31 or 32, wherein The disease associated with neuronal damage or neuronal death is selected from Parkinson's disease, visual system diseases related to loss of function or death of RGC or photoreceptor cells, Alzheimer's disease, brain injury, Huntington's disease, epilepsy, depression, sleep disorders, cerebral ischemia, motor neuron disease, amyotrophic lateral sclerosis, spinal muscular atrophy, ataxia, PolyQ disease, schizophrenia, addiction, Pick's disease, blindness, and deafness; preferably, Parkinson's disease and visual system diseases related to loss of function or death of RGC or photoreceptor cells.

35. The use according to claim 34, wherein The visual system disease associated with loss of function or death of RGC or photoreceptor cells is preferably selected from: vision impairment caused by death of RGC cells or photoreceptor cells, glaucoma, age-related RGC lesions, optic nerve damage, age-related macular degeneration (AMD), diabetic retinopathy, retinal ischemia or hemorrhage, Leber hereditary optic neuropathy, or a combination thereof; the visual system disease associated with loss of function or death of photoreceptor cells is preferably selected from: degeneration or death of photoreceptor cells caused by damage or degenerative lesions, macular degeneration, retinitis pigmentosa, diabetes-related blindness, night blindness, color blindness, hereditary blindness, congenital amaurosis, or a combination thereof.

36. The use according to claim 31 or 32, wherein The neurons are dopaminergic neurons, 5-HT neurons, NE neurons, ChAT neurons, GABA neurons, glutamatergic neurons, motor neurons, photoreceptor cells (such as rod cells and cone cells), retinal ganglion cells (RGC), cochlear nerve cells (such as cochlear spiral ganglion cells and vestibular neurons), or medium spiny neurons (MSN) or a combination thereof, preferably dopaminergic neurons, retinal ganglion cells and photoreceptor cells.

37. A method for preventing or treating a disease associated with neuronal damage or neuronal death, comprising administering to a subject in need thereof an effective amount of the negative regulator or positive regulator according to any one of claims 1 to 30.

38. The method according to claim 37, wherein The disease associated with neuronal damage or neuronal death is selected from Parkinson's disease, visual system diseases related to loss of function or death of RGC or photoreceptor cells, Alzheimer's disease, brain injury, Huntington's disease, epilepsy, depression, sleep disorders, cerebral ischemia, motor neuron disease, amyotrophic lateral sclerosis, spinal muscular atrophy, ataxia, PolyQ disease, schizophrenia, addiction, Pick's disease, blindness, and deafness; preferably, Parkinson's disease and visual system diseases related to loss of function or death of RGC or photoreceptor cells.

39. The method of claim 38, wherein The visual system disease associated with RGC dysfunction or death is preferably selected from: vision impairment caused by RGC cell death, glaucoma, age-related RGC lesions, optic nerve damage, age-related macular degeneration (AMD), diabetic retinopathy, retinal ischemia or hemorrhage, Leber hereditary optic neuropathy, or a combination thereof; the visual system disease associated with photoreceptor cell dysfunction or death is preferably selected from: photoreceptor cell degeneration or death caused by damage or degenerative lesions, macular degeneration, retinitis pigmentosa, diabetes-related blindness, night blindness, color blindness, hereditary blindness, congenital amaurosis, or a combination thereof.

40. The method according to claim 37, wherein The neurons are dopaminergic neurons, 5-HT neurons, NE neurons, ChAT neurons, GABA neurons, glutamatergic neurons, motor neurons, photoreceptor cells (such as rod cells and cone cells), retinal ganglion cells (RGC), cochlear nerve cells (such as cochlear spiral ganglion cells and vestibular neurons), or medium spiny neurons (MSN) or a combination thereof, preferably dopaminergic neurons, retinal ganglion cells and photoreceptor cells.

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